I recently read the article Threshold for Positivity and Optimal Dipyrone Concentration in Flow Cytometry-Assisted Basophil Activation Test by Hagau et al., in the August 2013 issue of AAIR, where the authors assessed the usefulness of basophil activation tests (BATs) in the diagnosis of allergy to dipyrone.1 Allergy to dipyrone still represents a main concern and pyrazolones are considered a major cause of immediate IgE-mediated reactions to drugs in many countries.2 A case of drug rash with eosinophilia and systemic symptomatology (DRESS) attributed to dipyrone was recently reported; however, the authors could not rule out a possible interfering role attributed to herbal mixtures and other drugs.3 BATs represent an interesting tool to diagnose drug hypersensitivity, though with some criticism about their performance and interpretation.4 Basophils captured in flow cytometry by CD193 (CCR3)-phycoerythrin (PE) may be contaminated by CD3⁺ expressing cells, leading to possible bias.5 Despite this issue, Hagau and colleagues reported that commercial BAT equipped with a Flow2-CAST technique, showed a sensitivity as low as 0.25 µg/mL dipyrone, about 3 orders of magnitude lower than previous reported evidence.1,2 In the paper by Gomez et al., a commercial BAT using an anti-IgE/CD63 protocol (instead of a CCR3/CD63) reported that the lowest dose able to trigger CD63 upregulation over the 5% threshold, was 0.25 mg/mL dipyrone (matamizole); Hagau and co-workers used the same CD63% threshold.1 It apppears quite difficult to attribute this sensitivity improvement to superior BAT performance (at least in regards to the phenotyping protocol).5 CCR3 has been recently reviewed as a phenotyping marker and compared to other flow cytometry approaches.5 The eotaxin receptor is commonly expressed on eosinophils,6 together with CD63 (which is only present on activated cells)7; however, in a SSC (side scatter)/CCR3 scatter plot, eosinophils and basophils can be clearly separated and eosinophils should not affect a Flow2-CAST BAT performance. Yet CCR3 is downregulated during basophil activation5 and the proportion of gated basophils evaluated as up-regulating CD63 is higher than the same calculated on resting, non activated basophils, if gate is set at a fixed threshold (usually ≤5%). In a SSC/CCR3 gating plot basophils may be underestimated, due to CCR3 downregulation and contamination by SSC^{low scattered}/CD3⁺ lymphocytes. If we consider that CCR3-PE^bright basophils express homogeneously very low amount of membrane CD63 (for example 5%), a supposed reduction of 52% of 500 gated CCR3-PE^bright cells (due to activation), might shift the threshold to 1.53%. This would mean that a proportion of CD63^neg cells enter the right side of the threshold and lead to a higher CD63% evaluation. The bias may be significant when CD63% is close to the threshold. In the paper, 1 SI is calculated on the number of basophils and Table 11 reports 6/20 samples (30%) with 5.3< SI <9.1. The simultaneous evaluation of CD63-FITC_MFI and CD63% should prevent a bias4; however, other bias can still occur. The operator might capture more CCR3-PE^bright cells than the negative control: in this case, as CCR3-PE^bright basophils are negative, the MFI ratio CD63^pos/CD63^neg may decrease while CD63% increase. In the lack of a CD3 marker, the operator may capture low SSC cells that express CCR3 but not CD63: this occurrence may also decrease the MFI ratio CD63^neg/CD63^pos and increase CD63%. Lowest doses of dipyrone may show a low expression of CD63-FITC (as median MFI) but relatively higher CD63% expressing basophils. This may prompt researchers to evaluate BAT as a valuable tool to probe very low doses of dipyrone in allergy; however, the absence of a flow cytometry plot reported by the authors1 makes it difficult to address this point and to elucidate possible causes about the high sensitivity reported.