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

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Amy Carpenter Aquino

December 2018—In allergy testing, microarray technology offers speed and the benefit of smaller sample volumes, but it has a lower sensitivity and is unable to detect IgE antibodies of all specificities in a given extract unless all allergens are on the chip. For routine use, singleplex assays are here to stay.

“Microarray technology will remain a wonderful research tool but will probably not emerge into the clinical world in a serious way for a variety of reasons,” among them a lack of FDA approval, said Robert G. Hamilton, PhD, D.ABMLI, director of the Dermatology, Allergy, and Clinical Immunology Reference Laboratory at Johns Hopkins University School of Medicine.

Dr. Hamilton, who is also a professor of medicine and of pathology at Johns Hopkins, shared a “global perspective of where microarray technology fits into the grand scheme of things,” with its pros and cons, at this year’s American Association for Clinical Chemistry annual meeting.

“The emergence of recombinant components drove us to consider using microarray technology,” he said.

The first allergen chip was developed in 2000 with recombinant allergenic proteins. In 2007, Thermo Fisher Scientific released the primary microarray, the ImmunoCAP immuno solid-phase allergen chip (ISAC), which can test 112 antibody specificities in a single serum analysis. “There have been several publications reporting expanded chip assays with 170 allergenic components and 282 allergenic components and extracts,” he said (Lupinek C, et al. Methods. 2014;66[1]:106–119; Heffler E, et al. World Allergy Organ J. 2018;11:7).

Dr. Hamilton

Microarray technology is a recent addition to the established “top down” strategy for diagnostic testing, which starts with the patient’s clinical history and examination and is followed by a skin prick test or serological IgE antibody analysis to confirm the specificity of the IgE antibody being present, Dr. Hamilton said. “And as we know, IgE is necessary but not sufficient for eliciting an allergic response and thus generating a definitive diagnosis of allergic disease.”

Allergenic components constitute the second phase in serological testing. “Reflexing to the allergenic components after the specific IgE test with actual extracts is positive is commonplace today,” he said.

While there are pros and cons to the use of allergen extracts, “the bottom-line conclusion is that we will remain using extract-based, solid-phase allergosorbents in IgE antibody serology as a primary tool and expand it using components where it is clinically useful,” Dr. Hamilton said.

Allergen extracts are relatively easy to prepare, and they provide the most comprehensive information for confirmation of sensitization. “They’re physiological extracts and they should contain a comprehensive amount of the allergenic components in that substance specificity.”

“The problem is that they are very heterogeneous and their potency and specificities can sometimes vary between lots and manufacturers,” Dr. Hamilton said. Even global warming is causing changes in some allergen extracts. The level of Amb a 1 in ragweed extracts is increasing, for example, because of rising temperatures and CO2 levels, which affect the amount of allergenic protein in the pollen that is generated.

Extracts also are highly variable, and there is little quality control. Nineteen of roughly 1,000 extracts used in vivo for diagnosis and immunotherapy are standardized. “Standardized is a soft term when it comes to an extract,” Dr. Hamilton said, “even when the level of an allergenic component is measured in the extract—like Fel d 1 in cat—or a biological allergy unit is assigned based on a biologic response in multiple concentrations of the extract placed on the skin of allergic people.”

Allergenic extracts do not lend themselves to easy differentiation of primary sensitization from a cross-reactivity–driven response because of the complexity of the extract itself, he said. “And you can never predict the relative risk of having a systemic allergic reaction using an extract-based diagnostic test.” Peanut is an example of a case in which reflexing to a group of components (Ara h 1, 2, 3, 6, 8, and 9) makes it possible to form assumptions about the potential risk for a serious reaction in peanut allergic individuals who encounter peanut exposure.

“Diagnostic allergen extracts aren’t perfect but they’re not obsolete,” Dr. Hamilton said. “Running a single allergen extract, let’s say peanut, where in theory it contains most of the allergenic components that are extractable from a peanut, is a good initial test for assessing sensitization in an individual.”

The main strength of the allergen extract is that “it’s a more comprehensive presence of all the allergenic components in that particular specificity versus running a single allergen, where we have to run all the single allergens that are available to cover the one specificity of interest.”

A comparison of allergen extracts and molecular allergens in the areas of purity, potency, availability, quality control, processing artifacts, and comprehensive nature (see table) shows the advantages of molecular allergens. For example, increased regulation of in vivo allergen extracts is limiting their availability while enhanced technology is expanding access to molecular allergens. “Jay Slater’s group at the FDA is identifying a subset of diagnostic and therapeutic allergenic extracts that are being removed because there’s no definitive evidence in the literature that they’re actually efficacious,” Dr. Hamilton said.
One reason for using molecular allergens in IgE antibody diagnostics is concern with the low abundance and/or weak stability of select allergenic molecules. “The illustrative case for me was when we were measuring IgE antibody to hazelnut and all of a sudden it went from very low levels to very high levels with the same specimen,” Dr. Hamilton said.

The manufacturer supplemented the hazelnut extract with Cor a 1, the group 1 allergen in hazelnut that is poorly extracted in a physiological buffer. Recombinant Cor a 1 was added to the hazelnut extract without widely advising customers. “That was the problem,” Dr. Hamilton said. “Our allergists were concerned because all of a sudden they were getting extremely high levels of hazelnut-specific IgE.”

Since Cor a 1 is cross-reactive with Bet v 1, a 17-kilodalton protein from birch pollen, the birch pollen allergic individuals were causing cross-reactive IgE antibody to bind to the hazelnut extract allergosorbent that had been supplemented with Cor a 1. Other examples are adding Gly m 4 in the extract in soy, and omega 5 gliadin in the wheat extract.

“You can take recombinant components and actually supplement the extract and make it a better extract by addressing the concern of the low abundance of certain allergens that are labile,” he said.

Molecular allergens make it possible to better assess the relative risk of selected allergen exposures. “The illustrative case is peanut, where storage proteins Ara h 1, 2, and 3—with Ara h 2 being the best indicator—allow us to make a better assumption that there is a higher risk of having a systemic reaction, versus dealing with sensitization to cross-reactive allergens such as Ara h 8 and 9.”

Cross-reactivity, too, can be assessed. “If we measured Bet v 1 specific IgE in birch pollen allergic individuals, we can expect there to be a broad cross-reactivity with IgE antibody to structurally similar molecules in the PR10 family of proteins,” Dr. Hamilton said.

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