1. A flow cytometry–based diagnosis of eosinophilic esophagitis
Ting Wen, PhD, Jonathan Kuhl, BS, Philip Putnam, MD, Vincent Mukkada, MD, Michael Farrell, MD, Ajay Kaul, MD, Conrad Cole, MD, Marc E. Rothenberg, MD, PhD 
Journal of Allergy and Clinical Immunology 
Volume 140, Issue 6, Pages e3 (December 2017)
DOI: /j.jaci
Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
FACS-based methodology recognizing major leukocyte populations present in human esophageal biopsy specimens. A, Schematic illustration of the diagnostic method. The biopsy tissue is digested into a single-cell suspension and then subjected to multicolor fluorescent antibody staining and FACS analysis, enumerating the number of eosinophils, mast cells, and basophils present in the digested biopsy specimen and resulting in a summation score for the diagnosis of active EoE. B, An active EoE biopsy specimen (50 eosinophils/HPF) was stained for CD45, CD117, CD11b, FcεRI, and Siglec-8. Representative plots are shown with a biopsy specimen from a patient with active EoE. Gated CD45+ events contain eosinophils (EOS; red), mast cells (MC; purple), and basophils (BASO; light blue). In the red frame the events presented in the left panel were plotted in the context of the eosinophil markers Siglec-8 and CD11b. A control sample (0 eosinophils/HPF) was juxtaposed for comparison purposes. C, The number of total single cells (singlets) was enumerated in biopsy specimens from patients with active EoE and control biopsy specimens. D, The number of CD45+ singlets was plotted for biopsy specimens from patients with active EoE versus control biopsy specimens. E, Tissue IELs were enumerated for biopsy specimens from patients with active EoE versus control biopsy specimens. F, Percentages of IELs of total CD45+ leukocytes were plotted for biopsy specimens from patients with active EoE versus control biopsy specimens. Scatter plots in Fig 1, C-F, are presented as means ± SEMs. **P < .01 and ***P < .001, 2-tailed Mann-Whitney or t test. Active, Active EoE.
Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci )
Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Diagnostic merit of the FACS-based method in reference to pathology. A, With FACS enumeration of a single esophageal biopsy specimen, the number of eosinophils (EOS), mast cells (MC), and basophils (BASO) was plotted. B, left panel, An EMB score was derived through summation of the absolute numbers of eosinophils, mast cells, and basophils as enumerated by using FACS, with red-labeled dots representing patients with GERD. Dashed line: The diagnostic cutoff of 285 EMB units was derived by means of receiver operating characteristic (ROC) analysis (right panel), resulting in an ROC curve with an area under the curve (AUC) equal to C, Number of eosinophils enumerated by using FACS correlates with eosinophil counts by using the standard-of-care histologic method (P < .0001, Spearman r = 0.78). D, Disease status scoring (left panel) and diagnostic merit (ROC, right panel) of using eosinophil enumeration only, with red-labeled dots representing patients with GERD. Data in Fig 2, A, B, and D are presented as means ± SEMs. **P < .01 and ***P < .001, 2-tailed Mann-Whitney test. E and F, EMB score and eosinophil enumeration data were analyzed in light of the GERD factor, respectively. Data in Fig 2, E and F, are presented as means ± SEMs. **P < .01 and ***P < .001 on EoE factor 2-way ANOVA. Active, Active EoE.
Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci )
Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig E1
FACS diagnosis with samples from patients with active EoE (Active) and control samples with less than 2 eosinophils/HPF. The active EoE cohort and a subcohort of the control samples with less than 2 eosinophils/HPF were included in the diagnostic merit assessment to mimic the diagnostic need to distinguish inactive control samples and samples from patients with EoE. The EMB score was calculated on the basis of the previously introduced EMB algorithm (left panel), and receiver operating characteristic (ROC) analysis was performed between the control subcohort and the active EoE cohort (right panel). Data are presented as means ± SEMs. AUC, Area under curve.
Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci )
Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions