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McGill Team Devises Streamlined, Inexpensive Microarray IHC Method

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NEW YORK (360Dx) – Researchers at McGill University have developed a microfluidic immunohistochemistry assay that enables multiplexed protein detection while retaining sample structural and spatial information.

The group has launched a spinout, Parallex BioAssays, to commercialize the underlying technology, which uses slides preprinted with antibodies to target proteins to enable what Huiyan Li, a postdoc in the lab of McGill professor David Juncker, said is a streamlined and inexpensive approach to multiplexed IHC.

In a paper published last week in Analytical Chemistry, the researchers used the approach to measure levels of eight proteins in a breast cancer tissue section using a total of 180 antibody spots. The technique could ultimately allow for more highly multiplexed clinical pathology assays, Li said, but, more immediately, could prove a useful platform for discovery and validation of tissue biomarkers.

Protein IHC is one of the primary tools pathologists use to provide diagnoses and prognoses in cancer and a variety of diseases. Conventional IHC typically looks at one protein at a time. However, as Li noted, given the complexity of conditions like cancer, the ability to look at multiple proteins could prove clinically valuable.

Methods like reverse phase protein arrays (RPPA) and tissue microarrays allow for multiplexed protein measurements, but both have downsides, particularly in terms of retaining the spatial and structural information contained in a conventional pathology slide.

For instance, Li said, tissue microarrays typically consist of small subsections cut from the larger patient tissue, which means some of the spatial information is lost. In RPPA, spatial information is destroyed when the tissue is made into a lysate that can be spotted on the slide.

In the McGill approach the researchers "keep the whole tissue section but use different antibodies in different sections of the tissue, which keeps the spatial information," Li said.

As Li and her coauthors noted, other groups have developed similar methods for multiplexing proteins on intact pathology samples, but, she said, these approaches have typically required equipment like syringe pumps and pressure controllers that are expensive and not typically present in pathology labs.

The McGill researchers simplified this process by applying the "snap-chip" technology also developed in Juncker's lab. This technology allows researchers to transfer prespotted drops of reagents (in this case antibodies to the target proteins) from one microarray slide to another. In the microarray IHC assay, antibodies are prespotted on a slide and then transferred via the snap-chip device to the tissue slide. This means the antibody slides can be preprinted at a facility with the required printing equipment and then sent to the pathology or research lab where they can be applied to the tissue of interest.

Parallex BioAssays has commercialized the snap-chip technology and offers assays multiplexing up to 100 antibodies or assay conditions per chip. Based in St-Basile-le-Grand, Quebec, the company was founded by Sebastien Bergeron, formerly a researcher in Juncker's lab and the coauthor on several studies describing the technology.

As a proof of principle, the researchers used the method to measure eight proteins — the breast cancer subtype markers ER, PR, and HER2, along with five cancer-related proteins — in a formalin-fixed, paraffin-embedded breast tumor slide. The tumor subtype was ER-, PR+, HER2-, and consistent with this, the assay found elevated levels of PR and average expression of HER2 and ER. They also found overexpression of P53 and PTEN, which they noted is likewise consistent with previous analyses of this breast cancer subtype.

In addition to streamlining the microarray IHC approach, the method also enabled an increase in array density by two orders of magnitude compared to previous efforts, the authors wrote, allowing a piece of tissue on the order of 25 mm2 to be stained with 100 microspots. Reagent costs for the assay described in the study came to around $.20 per target.

Li noted that one key consideration as she and her colleagues continue development of the method is determining the required spot density for particular proteins in particular samples. Because the microarray IHC approach stains not the entire tissue section but samples different spots within the section, tumor heterogeneity presents a potential issue.

Answering this question will require "a relatively large [number] of samples to test and experimentally determine how many spots we need for each protein," Li said, noting that the required density will depend in part on how accurate a measurement is needed of a given biomarker to make a clinical decision. "We'll have to do many replicates to get confident results on that question."

Most important, she noted, is establishing the density required to avoid false negative diagnoses so that the technique does not miss cancer cases.

The researchers are currently working with pathologists at McGill University Health Center to collect samples for this work and to establish, more generally, that their results match up with those returned by conventional pathology.

"We are working on breast cancer mainly, and so now, we need to collect different samples from different ages of patients and different subtypes and study different biomarkers to see how many spots we need [to get good results]," Li said.

She added that she believes the method "has great potential to make it into the clinical lab in the future," given that it is simple to use and relatively inexpensive. In the nearer term, she suggested the approach would prove useful for discovery and validation of tissue-based markers.

Li said she and her colleagues are also looking to apply the method to other cancers and diseases. "It can be useful really for any disease that requires multiplexed protein detection," she said.