2016 Section 5 Green Book

A total of 100 l L aliquots of cells were distributed to poly- styrene fluorescent activated cell sorter (FACS) tubes at room temperature, in the dark, for 30 minutes. Temperature single- cell suspensions were stained for CD3, CD4, CD8, CD19, CD45, and CD56 using antihuman fluorochrome conjugated antibodies and incubated at 4 C for 30 minutes according to established BD Biosciences protocols. Following the extracellular stain, 250 l L of BD Cytofix/Cytoperm fixation and permeabilization solu- tion (BD Biosciences) was added to each sample for 20 minutes at 4 C in the dark. Intracellular cytokines were stained for IL4, IL5, IL13, IL17, and interferon (IFN)- c using specific antibodies or appro- priate isotype controls for 30 minutes at 4 C in 1 3 BD perm/ wash buffer (BD Biosciences). Cells were washed twice with perm/wash buffer according to the manufacturer’s instructions (BD Biosciences). Cells were fixed with a preparation of 2% paraformaldehyde (PFA v/v in phosphate-buffered saline) and stored at 4 C covered in the dark. Samples were run through a Cytek 8DXP upgraded (Cytek Development, Fremont, CA) FACSCalibur (BD Biosciences). Flow cytometer and fluorescence data were acquired using FlowJo software version 4.6 (TreeStar, Ashland, OR). Gates were created based on isotypes and fluorescence-minus-1 con- trols. For the intracellular cytokines, positive signal for CD45 was used to gate on polyp leukocytes. T helper cells were sub- gated from the leukocyte population using CD4 antibody. The resulting CD4 1 leukocytes were then analyzed for IL4, IL5, IL13, IL17, and IFN- c –producing cells. Intracellular cytokines were also analyzed for CD45 1 CD4 2 s cells. Baseline significance for this study was set at a 5 .05. All groups were compared using ANOVA on SPSS statistical soft- ware (IBM SPSS) with appropriate Tukey post hoc analyses. In some analyses, Student t tests with Bonferroni-Holme correc- tions were conducted.

Fig. 5. Microscopic hematoxylin and eosin slide (2 3 ) of a nasal polyp demonstrating superficial subepithelial distribution of cells within the rectangle.

stromal (Fig. 4) or subepithelial space distribution (Fig. 5). The inflammatory cells in this dense area were counted to a total of 500 cells or until all inflammatory cells (EO, PMN, lymphocyte, plasma cells) in the polyp sample were counted. Data were nor- malized as a ratio to the total cell count. To determine the cellularity of the polyp sample, five consecutive high power fields (HPFs) (1,000 3 ) were used to count the total number of EO, PMN, lymphocyte, and plasma cells in one HPF. The aver- age number of the total cell count from the five HPFs of each patient was defined as the cellularity of the nasal polyp. Ten HPFs (1,000 3 ) of the epithelium were analyzed to characterize and count the goblet cells present. The surface epi- thelial morphology was categorized as either pseudostratified ciliated columnar or transitional epithelium. The area of high- est concentration of mast cells was identified. Ten consecutive HPFs (400 3 ) were used to count for mast cells. The average number of mast cells for each patient was obtained. Data analysis was performed using analysis of variance (ANOVA) on SPSS statistical software version 17.0 (IBM SPSS, Armonk, NY) with appropriate Tukey post hoc analyses. P val- ues < .05 were considered to be statistically significant. Flow Cytometry The same polyp specimen used in the histologic examina- tion was used for flow cytometry. Fresh tissue specimens were placed in Royal Park Memorial Institute (RPMI) 1640 1 3 me- dium (Cellgro, Manassas, VA) and processed within 1 hour of extraction. Under a category 2 sterile hood, tissue samples were disaggregated to allow separation of cells from the tissue. Cell suspensions were prepared from the resulting eluent, using a 70 l m BD Falcon cell strainer (BD Biosciences, Franklin Lakes, NJ). Pelleted cells (400 3 g , 4 C, 5 minutes) were stimulated in nonpolarizing stimulation media to facilitate production of in- tracellular cytokines. Results were achieved for leukocytes by reconstituting cells in 1 mL RPMI 1640 supplemented with 10% fetal bovine serum (Lonza Group, Ltd., Basle, Switzerland), 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA), 1 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO), 0.6 l L BD Golgi stop protein transport inhibitor (BD Biosciences), and 500 ng/mL ionomycin (Sigma-Aldrich) and cultured for 5 hours at 37 C according to established BD Biosciences protocols.

RESULTS Phenotype

Eighty-four patients were included in the study, with ages ranging from 7 to 83 years of age (median age, 46 years) (Table II). CF was the youngest subclass and statistically lower than each asthmatic sinusitis group ( P < .01). There were 48 females in the study, with signifi- cant higher females in the asthmatic sinusitis (AScA

TABLE II. The Demographic Information for the CRS Subclasses.

CRS Subclass (n)

Mean Age (yr)

Female (n)

AERD (9)

46 34

5 4

AFS (11)

CF (7)

16

5

AScA (13)

48 35

11

ASsA (5)

4

NAScA (14)

54

7

NASsA (12)

59 50

3 9

Control (13)

Total (84)

46

48

AERD 5 aspirin exacerbated respiratory disease also known as aspirin triad; AFS 5 allergic fungal sinusitis; AScA 5 asthmatic sinusitis with allergy; ASsA 5 asthmatic sinusitis without allergy; CF 5 cystic fibrosis; CRS 5 chronic rhinosinusitis; NAScA 5 nonasthmatic sinusitis with allergy; NAS- sA 5 nonasthmatic sinusitis without allergy.

Laryngoscope 123: March 2013

Han: Subclassification of Chronic Sinusitis

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