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Multiplex for allergy dx: powerful, but it has its place

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Finally, molecular allergens are better biomarkers of species-specific sensitization. In hazelnut, for example, Cor a 19 and 14 are much more specific than using hazelnut extract itself on the allergosorbent in terms of assessing the presence of hazelnut sensitivity. Among the primary allergenic families of molecular allergens, Bet v 1 is particularly interesting, he said, citing work by Heimo Breiteneder, PhD, of the Medical University of Vienna. “If you have an IgE antibody to birch or Bet v 1, you can have IgE antibody that binds to structurally similar molecules in a variety of fruit, vegetable, legume, and nut specificities, and it’s tough to distinguish that without actually assessing it at a molecular level.”

Technology for detecting IgE antibody has evolved from isotopic to non-isotopic, from polyclonal to monoclonal anti-IgE, and to the use of heterologous interpolation from a total serum IgE calibration curve. New solid-phase matrix materials have become available with higher binding capacities, so “we’ve moved from the paper disks to the cellulose matrix,” Dr. Hamilton said. Robotics and automation have led to greater assay precision and reproducibility. “We’ve moved from extracts to molecular components on the allergosorbents, and we have seen the emergence of the multiplex technology.”

The ImmunoCAP ISAC chip is the predominant IgE antibody multiplex assay. The chip is a glass slide with four sub-squares each containing 112 individual allergenic components, spotted in triplicate, with controls for IgE to ensure the serum is added to the chip.

The assay itself is a standard solid-phase, fluorescent immunoassay where IgE binds with solid-phase allergen. A microarray reader scans the chip and produces an image, which is analyzed and interpolated into a report. Units are measured in semiquantitative ISAC standardized units, or ISUs. “It’s very similar in nature to a singleplex assay, except that you’re simultaneously analyzing IgE antibody to multiple allergens on the actual assay.”

FDA approval is one of the barriers to multiplex analysis, Dr. Hamilton said. “You won’t find chip-based microarrays being run as diagnostic tests in clinical laboratories. They’re great research tools. We use them in a variety of epidemiologic studies, and they are a powerful technology for examining cross-reactivity, but they haven’t arrived yet at federal clearance as a diagnostic test.”

He added: “How would the FDA verify the quality control of 112 individual allergens on a chip? They’re currently exploring ways to validate the performance of even one or two allergens on a chip.”

One of the main strengths of allergen component-based multiplex analysis is its superior ability to identify IgE anti-food/pollen allergen cross-reactivity. It can also detect the presence of relevant IgG- and IgA-blocking antibodies. “Because of the limited amount of allergen on the chip, the IgG antibodies that are present can compete for binding sites with IgE, and this causes an artifactual reduction in the detectable level of IgE antibody,” Dr. Hamilton said. Viennese researchers have suggested the positive aspect of this interference, which is the potential in using the multiplex assay as a tool to examine the effects of immunotherapy where a patient is building up IgG blocking antibody.

The weaknesses of allergen component-based multiplex analysis currently outnumber its strengths: The analytical sensitivity is lower than with singleplex assays, it will not detect IgE antibodies of all specificities in a given allergen extract unless all allergens are on the chip, and IgE binding can be interfered with by antibodies of the same specificity but different isotypes (“a real negative, in my opinion, in many ways,” he said).

“Most importantly for me is what we preach when we’re teaching the allergists in terms of practice of ordering tests: You order the specificities in which you suspect the individual patient is clinically sensitive, or at least indicates based on a positive history that they are sensitive,” Dr. Hamilton said. “You don’t run 100 allergen specificities as a fixed panel at one time. It’s very inefficient, and you’re producing data you then may have to explain away, because the individual is IgE antibody positive for a particular specificity but has no positive history relevant to that specificity.”

“This panel-testing concept is a concern,” he added. “You can look at it as both a strength and a weakness. It unfortunately encourages abuse of testing.”
The singleplex assay offers performance-related advantages: greater analytical sensitivity, more precise quantification and precision, and established internal and external quality control measures. “It’s much more rigorous versus the multiplex assay’s strengths, which are speed and conservation of the sample, running 30 to 40 microliters to measure 112 individual allergenic specificities.”

The singleplex assay uses a calibration system that is traceable to the third WHO International Standard for serum IgE and has similar units across various assay methods. “Within any given assay, however, it is remarkably reproducible,” he noted. “You can get the same result here in Chicago that you can get in Tokyo because of the reproducibility and stability of the reagents that are used worldwide.”

It also minimizes unneeded testing, is available worldwide, and is more cost efficient in the case of a limited sample number. But the greater need for reagents makes it more costly to run and it requires more technical intervention by staff.

In contrast, the multiplex assay requires fewer reagents and less technician intervention. With its speed and smaller sample volumes, especially beneficial in pediatric testing, “it’s ideal as a point-of-care test.”

A POC assay was on the market briefly. The ImmunoCAP Rapid is a lateral-flow assay that was FDA cleared for 10 aeroallergen specificities. “The assay itself worked well, but the results were being interpreted by the individual patients or the mother and it was a problem,” Dr. Hamilton said. Allergists were concerned, he said, and “laboratories that offered more sophisticated assay testing would miss these additional test requests, but it was a procedure for obtaining a diagnosis of sensitization that was being self-interpreted. Eventually this led to it being removed by the manufacturer from the market.”

Abionic’s IVD Capsule Aeroallergen is a nanotechnology biosensor POC test that mixes serum with fluorescent anti-IgE. The mixture is added to a capsule containing 10 allergens coupled with biosensors on the biosensor surface. Capillary action drives the IgE to immobilize the allergen, and the fluorescent result is measured optically. “Its limitation is the number of specificities, but it’s a novel test.”

Among the proof-of-concept assays is a hydrogel biochip in which 21 allergens, 15 extracts, and six molecules are embedded in gel. The chip has a limited binding capacity, Dr. Hamilton said, and there is limited information about what is binding to the chip.

The multiplex Luminex xMAP-based microarray, designed for indoor aeroallergens, uses an anti-allergen complex on a solid phase. “The assay itself performs well but it’s rather limited,” he said.

Multiplexing systems or reflex singleplex assays using allergenic components offer the greatest potential for natural history and epidemiological studies and for mapping geographically aeroallergen exposure and sensitization patterns. A study published in 2017 examined the geographic distribution of peanut and peanut component specific IgE in a large U.S. population (Valcour A, et al. Ann Allergy Asthma Immunol. 119[3]:262–266.e1).

The complex allergen extract has obvious imperfections but “it’s going to be with us for a long time,” Dr. Hamilton said. The challenge is to consider all the patient and environmental factors that drive the IgE antibody response and look at the four parameters of the immune response that are clinically important: concentration of IgE, affinity, clonality, and specific activity (specific IgE to total IgE ratio). “How many allergists actually measure the total IgE and look at the specific IgE in relation to the total IgE?” he said, noting that this specific activity is a clinically important but overlooked parameter of the immune response.

Whatever the method selected, whether a single-, oligo-, or multiplex assay, “interpretation must be guided by and viewed within the context of the patient’s clinical history,” Dr. Hamilton emphasizes. And he repeats: “The presence of IgE antibody sensitization is necessary but not sufficient for making the definitive diagnosis of human allergic disease.”

Amy Carpenter Aquino is CAP TODAY senior editor. For additional coverage of the AACC session on allergy testing, see “Component IgE testing offers food for thought,” CAP TODAY, November 2018, www.captodayonline.com.

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