Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance Lara S. Ford, MD, MPH, Katherine A. Bloom, MD,

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Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance Lara S. Ford, MD, MPH, Katherine A. Bloom, MD, Anna H. Nowak-Węgrzyn, MD, Wayne G. Shreffler, MD, PhD, Madhan Masilamani, PhD, Hugh A. Sampson, MD Journal of Allergy and Clinical Immunology Volume 131, Issue 1, Pages 180-186.e3 (January 2013) DOI: 10.1016/j.jaci.2012.06.003 Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Casein-specific IgE measurements by 5 levels of challenge outcome. With the 3 central groups pooled, post hoc analysis showed a significant difference between those who reacted to baked milk and those who tolerated it (P < .001) and between those who tolerated baked milk and those who had fully outgrown their milk allergy (P < .005). BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Casein-specific IgG4 measurements by 5 levels of challenge outcome. BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Casein-specific IgE/IgG4 ratios by 5 levels of challenge outcome. With the 3 central groups pooled, post hoc analysis showed a significant difference between those who reacted to baked milk and those who tolerated it (P < .01) but not between those who tolerate baked milk and those who had fully outgrown their milk allergy (P = .25). BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Ratio of milk-specific to anti-IgE (nonspecific) basophil activation by 5 levels of challenge outcome. With the 3 central groups pooled, post hoc analysis showed a significant difference between those who reacted to baked milk and those who tolerated it (P < .05) but not between those who tolerate baked milk and those who had fully outgrown their milk allergy (P = .11). BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Mean fluorescence intensity (mfi) of CD203c after stimulation with RPMI (negative control) by 5 levels of challenge outcome. This measure forms an indication of spontaneous activation. BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Wheal reaction on SPTs with commercial milk extract by 3 levels of challenge outcome (baked milk–tolerant subjects have been pooled). Post hoc analysis shows a significant decrease in wheal size in those who tolerated baked milk compared with those who reacted to it (P < .005) and in those who have fully outgrown their milk allergy compared with those who tolerate baked milk (P < .005). BM, Baked milk; PST, skin prick test. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Receiver operating characteristic curves showing performance of various tests in predicting clinical reactivity to baked milk. Casein IgE has an area under the curve (AUC) of 0.78 (95% CI, 0.69-0.88), cow's milk IgE has an AUC of 0.73 (95% CI, 0.63-0.83), milk-specific basophil reactivity has an AUC of 0.69 (95% CI, 0.59-0.80), and milk skin prick test (PST) wheal diameter has an AUC of 0.68 (95% CI, 0.58-0.78). Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Example of basophil gating strategy. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; SSC, side scatter. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Basophil reactivity to milk by 5 levels of challenge outcome. With the 3 central groups pooled, post hoc analysis showed a significant difference between those who reacted to baked milk and those who tolerated it (P < .01) and also between those who tolerate baked milk and those who had fully outgrown their milk allergy (P < .05). BM, Baked milk; R.P., rice pudding. Journal of Allergy and Clinical Immunology 2013 131, 180-186.e3DOI: (10.1016/j.jaci.2012.06.003) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions