A paper by Ariza et al., published in the current issue of Cytometry Part A, described the ability of basophil activation test (BAT) in diagnosing patients with cross-intolerance to NSAIDs 1. Ariza et al. concluded that BAT used in their study did not appear useful to evaluate patients with NSAID-derived allergy, despite the fact that their flow cytometry approach to investigate basophils by a CD193/CD203c/CD63 BAT represented a novelty in the field. The data reported by the authors 1, particularly as for Table 1 of their paper, indicated that a main pitfall of BAT to evaluate NSAIDs allergy was the many false-positive results, reflected in the low specificity obtained, which may be explained by NSAIDs capacity to interact pharmacologically with basophils, inducing cell activation in both NSAID-intolerant and NSAID-tolerant subjects, although they showed that only one single drug appeared to be in contradiction with a cross-reacting NSAID-hypersensitivity 1. The authors tried to address the problem by adding CD203c marker in a previously standardized CD193/CD63 gating protocol. Basophil phenotyping markers, such as CD193 (CCR3) and CD203c, deserved major attention in recent years 2-4. They are used to capture basophils from whole peripheral blood samples and allow to investigate cell response to allergens or other molecules in the in vitro assay by flow cytometry. Many of these molecules are also expressed on the surface of activated basophils, therefore, some concern raises when they are indifferently used either as phenotyping or activation markers 5-7. Ariza et al. used the eotaxin receptor CCR3 (or CD193) and the basophil specific marker CD203c to gate basophils and evaluate their CD63 expression on patients with NSAIDs-induced uticaria/angioedema (NIUA) 1. CD203c was fundamentally included in the protocol because is a highly specific marker expressed only by basophils and, therefore, it can be used to selectively gate CD193-expressing cells as basophils 1. The problem of CCR3 (eotaxin receptor or CD193) regards its coexpression on eosinophils (which anyway gate on a different SSC scale quite far from basophils, although few CD63-expressing basophils may be captured in the area) and on T-cells, for which adding a CD3 markers was recently recommended 3. Despite to their heavy endowment of granules, basophils gate on low SSC within the lympho-area; and therefore, it is quite difficult to find these cells on high SSC where eosinophils are located in a flow cytometry (FC) dot plot. On the contrary, T cells may be present within the CCR3+ events indicated as basophils. Furthermore, CCR3-expressing and CD203c-bright cells change their fluorescence following an activation signal, as allergens do, causing possible bias in FC electronic capture. BAT remains a flow cytometry approach that may provide a reasonable alternative to traditional mediator release assays used to test cell participation in allergy mechanism, despite the many issues about suitability, sensitivity, and specificity, which are yet to be further elucidated 8. For certain aspects, practitioners may feel as staggering explorers over a fluttering icepack in the Arctic, when gaining full awareness of the many criticisms raised on the technical aspects with which most of BATs are performed and interpreted. The metaphor should be justified on the basis that BAT is a cellular test used with metrics usually pertaining to a serological assay. Complexity associated with this cell behavior during allergy should expand the debate how to solve the many pitfalls associated with BAT performance, then trying to enhance its diagnostic potential. For example, main weak point is represented by criteria used to determine the activation cut off and CD63% threshold. For serological markers, cut off can be easily calculated on the basis of quantitative evaluation, while setting a cut off for a cell response is a quite more puzzling approach. Spontaneously activated basophils exhibit a CD63 upregulation, which may encompass a CD63% range from 1.5 to 2.5% 9. The bimodal behavior od CD63 upregulation involves a significant range of CD63-expressing basophils which could be included in the right/positive area defined by the cut off threshold with a wide dispersed CD63-expressing cell population and a very little CD63-bright expressing more homogenous population (Fig. 1). The latter should represent those cells that upregulated CD63 efficiently following the allergen challenge. Usually, most of these basophils appear in a CD63 area higher than 10%. Papers considering a cut off >2–5% should exclude about 10% of basophils resulting CD63 negative probably due to anergic, nonresponding subjects 10. Table 1 shows that for a cut off ≥10% the percentage of positive diagnosed allergic subjects (sensitivity) is higher and often associated with a high specificity, for example, reaching values, respectively, of 92% and 80% with a 15% cut off threshold 21. By increasing cut off, one might even gain a good test performance without changing the methodological setting 21. Conversely, a relatively lowest cut off should ask for a better gating performance of BAT, by ameliorating FC capture and activation monitoring (see Table 1). Evaluation of dead cells by PI-annexin-V markers, calcium influx, membrane rafting or the many additional membrane and intracellular markers can optimize BATs 4, 8 and in this sense some author suggested also mean fluorescence (MFI) being included 23, 24 to ameliorate CD63-based BAT diagnostic potential. BAT-related diagnostic outcome depends also on the gating/assay approach used for the specific cellular assay. Although Ariza et al. set a CD63% cut off ≥5.0%, on the basis of previously reported evidence 1, other colleagues considered that the best cut off for activated basophils, usually established by analysing the receiver operating characteristic curves of results observed in a selected group of subjects with hypersensitivity reactions versus controls, could easily include values such as 2% 25, 6% 18, 13% 20, or 15% 21, as summarized in Table 1. Although setting a cut off value depends on the technical and analytical performance of the assay, besides to epidemiological components such as serum markers prevalence within tested population, the wide range here reported should ask for a Consensus Conference about the use and interpretation of BAT in allergy, to avoid arbitrary or even too simplistic approaches 8. A recent European Academy of Allergy and Clinical Immunology (EAACI) position paper for the diagnosis of urticaria reported that the use of different methodologies to gate basophils and evaluate activated cells, appears to affect the outcome of the assay 26. Although the EAACI report suggested a BAT protocol based on a CD123/HLADR gating strategy 26, 27 and the elimination of in vitro stimulation (e.g., with IL-3), the activation cut off should be yet calculated depending on the different BAT protocols used 5, 8, 26. The use of the priming cytokine IL-3 in BAT is a matter of debate 28 but it should not affect substantially this issue, as the only pitfall to which one could refer regards the fact that preanalytical handling procedures may increase IL-3-mediated response of basophils, but mainly on CD203c expression 9. Notwithstanding, this raises some concern for CD63 expressing cells considered positive at a cut off >5%, which may cause potential pitfall. Bias may arise from the consideration that positive CD63% close to a 5% cut off in the allergic population should account for active allergic individuals. A recent paper by Hagau et al., considered a threshold >5% for diagnostic criteria 29. This study showed BAT CD63% in 20 allergic patients elicited with neuromuscular blocking agents. All the investigated patients showed clear clinical symptoms of an allergic reaction. BAT CD63% mean value ±SD was 11.465 ± 7.85, accounting for 50% values higher than 10% CD63 and only 10% below or close to 5%. The latter values most probably reflect the existence of a nonresponder/anergic population of basophil, which was previously reported to generally occur in allergy 30. Therefore, there is still a gap between analytical in vitro evaluation, which considers a cut off >5% and clinical manifestations, where reliable BATs more frequently reach a CD63% ≥10%. The practitioner should consider that, at least 10–15% of allergic patients, may meet the nonresponder, anergic criteria 10, 30. Validation studies should be optimized by taking into account this consideration. Ariza et al. referred to an activation cut off of 5%, considering 2.5% of spontaneous CD63 activation, both values calculated on an anti-IgE-FITC + CD63-PE BAT protocol 31, while the same authors used the tricolor gating strategy CD193-APC/CD203c-PE + CD63-FITC in their paper 1. Despite this protocol review, results recently presented by Ariza et al., yet appeared to suffer from a moderate increase in CD63 membrane upregulation (see Fig. 1 in Ref. 1) and, moreover, positive values were significantly below those ones reported elsewhere, which resulted usually higher than 10–12% 21, 20. This fact may suggest that BAT diagnostic potential closely depends on cut off determination, but also on CD63 evaluation, whose activation threshold may be arbitrarily set. Only in this circumstance, preanalytical factors may enhance the bias 7. Actually, CD63 baseline level may be increased by IL-3, though in a more modest fashion than CD203c, even when released by cells following basophil preanalytical processing 9, an evidence that suggested the use of an ice-bath to reduce activation 28, therefore, CD63 baseline expression should be set adequately, prior to calculate the activation cut off during analysis 23. CD63 may be influenced by other CD63-expressing cells, such as platelets 25, its calculation depends on basophil responsiveness 23 and in a BAT using a stimulation buffer and CD203c as a gating marker, CD63 evaluation may be biased. A BAT using IL-3 to enhance the sensitivity of the assay can let IL-3 acting quickly, although its primary benefit in the short-time intervals used in these assays is to modify lipid mediator release. Usually, a slight enhancement of histamine release may be reported and there is some indication that it might modify the balance of piecemeal and anaphylactic degranulation 8, which therefore modifies the CD63 response. As CD203c responds to IL-3 alone, IL-3 pretreatment raises the apparent "no stimulus" CD203c expression, possibly blunting the stimulation index (SI) 8. These facts may occur during preanalytical handling of blood specimens. Activated basophils downregulate CD193 and upregulate CD203c, even in basal conditions. During allergic activation, the ratio CD203c/CD193 is higher as expected, possibly due to the concurrent decrease in CD193 expressing basophils paralleled to CD203c upregulation. This fact may explain why SI between CD193pos and CD193pos+CD203cpos in NIUA was significantly different than CD63%, expressed by different bar chart heights 1. These hallmarks would contribute to define the overall performance of a BAT and its reliability and suitability as a diagnostic tool for allergy. The paper by Ariza et al. is a well-designed report on the use of BAT but as they concluded that a CD193/CD203c + CD63 BAT did not fit as a reliable tool for NSAID-induced allergy, technical insights should be requested. Therapy of allergy and the study of allergic mechanisms may account on a wide variety of assays that in various ways assess the presence and function of IgE antibody. The study of allergic diseases could benefit from the study of basophils, as they are more accessible than mast cells for ex vivo studies. Flow cytometry investigation with BATs represents an outstanding opportunity to focus onto basophils and their involvment in innate immunity but even offers the ability to highlight those mechanisms able to optimize BAT as a reliable tool for the diagnosis of hypersensitivity reactions.
No takes yet. Share an insight, caveat, or question.
Salvatore Chirumbolo (2014) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: