Patients and biopsy samples
The study subjects were 46 patients with SSc (lcSSc 26, dcSSc 20, males 8, females 38) aged 60±12 years (mean±SD, range, 30–80 years). All patients met the 1980 American College of Rheumatology preliminary classification criteria for SSc (12). Patients with overlapping syndromes, mixed connective tissue disease, or other connective tissue diseases were excluded from the study. The clinical features of the 46 patients with SSc are listed in Table 1. The disease duration was 2.1±1.8 years, and the modified Rodnan’s skin thickness score (mRSS) was 9.2±9.1. Among the patients, 26% were positive for anti–topoisomerase I antibody and 32% were positive for anti-centromere antibody. Systolic blood pressure was 126±22.5 and diastolic blood pressure was 75±14.2.
Skin biopsy samples from patients with SSc (n=46) were obtained from areas of fingers and forearms with signs and/or symptoms of the disease. Skin biopsy samples from patients with non-SSc (connective tissue disease other than SSc) (n=29) served as control and samples from autopsy (n=10) served as normal controls. The non-SSc group and normal control group were age- and sex-matched to the patients with SSc, and consisted of 25 women and 4 men, and 6 women and 4 men, respectively. The mean±SD age of the control subjects was 56.0±15.9 years (range, 25-87 years) and that of normal control subjects was 68.1±11.9 years (range, 59–84 years). Each patient and control subject signed an informed consent form, and the study protocol was approved by the local institutional review board.

Clinical assessment
SSc was assessed clinically by mRSS, a clinical evaluation system of skin thickness in 17 body surface areas (face, chest, abdomen, right and left fingers, hands, forearms, upper arms, thighs, lower legs, and feet) (13). Each area was assessed for thickness using a 0–3 scale (0=normal, 1=mild but definite thickening, 2=moderate skin thickening, 3=severe skin thickening). The total score (the sum of scores from all 17 body areas) ranged from 0 to 51. Patients were classified into lcSSc and dcSSc based on the criteria of LeRoy et al. (14). Disease duration represented the time between onset of skin sclerosis and study entry.

Mast cells were detected by toluidine blue staining and c-Kit (CD117) immunohistochemical staining of biopsy samples. Briefly, skin biopsy specimens were taken with a disposable 5 mm biopsy punch from both the second finger and forearm, from an involved area in patients with non-SSc, or from a normal area from autopsy. The precise area, size, and depth of the skin biopsy were selected and performed by skilled dermatologists after consultation with the patient. Skin biopsy tissue sections were fixed in 4% formaldehyde for 1-2 days and embedded in paraffin wax. Sections of 5-6 mm thickness were cut, deparaffinized and stained with 0.05% toluidine blue (pH 4.0; Muto Pure Chemicals, Tokyo, Japan). The sections were then dehydrated and mounted in xylene-based medium for microscopic examination.
The NanoZoomer Digital Pathology (Hamamatsu Photonics K.K., Japan), a system that converts a histology slide into high-resolution digital slide (190 million pixels), was utilized for analysis. The NanoZoomer Digital Pathology enables sequential pathological analysis of multiple sections and accurate automatic measurement of the specimen area, namely, density and area of mast cells (/mm2) in each specimen, including those lying in the deep skin. The mast cell count was confirmed twice by two skilled pathologists who counted the cells manually. The average of the two counts was used in all calculations; the two results differed by <5%. In case of doubt, mast cells were re-examined at x1000 magnification using an oil immersion lens. Data are expressed as mean count per high-power field.

Studied tissues were cut to a thickness of 5mm, placed on glass slides coated with 3-triethoxysilyl-propylamine, deparaffinized in xylene, rehydrated in graded alcohols, and rinsed in distilled water. Sections were then treated with hydrogen peroxide in methanol (3%) for 30 minutes for inhibition of endogenous peroxidase. For antigen retrieval, citrate buffer (pH 6.0) was used for 15 minutes at 95°C in a water bath. Before application of the primary antibody, nonspecific binding was blocked for 1 hour with 10% normal goat serum in Tris-buffered saline (TBS). The primary antibody, CD117/c-Kit polyclonal rabbit anti-human (Nichirei, Tokyo), mouse monoclonal antibodies (MAbs) against human mast cell proteases, tryptase (Abcam, Cambridge, MA; AA1) and chymase (Abcam; SPM 195), was incubated for 30 min at room temperature, at a dilution of 1:100 in phosphate buffered saline (PBS). After washing, sections were incubated with secondary biotinylated anti-rabbit or anti-mouse immunoglobulin G (Histofine Simple Stain MAX PO, Nichirei) for 30 minutes at room temperature and then with the streptavidin–peroxidase complex for 25 minutes at room temperature. After incubation in DAB (diaminobenzidine 0.02% and H2O2 0.001% in PBS) for 12 minutes, sections were immediately rinsed with PBS and under running tap water, counterstained with Mayer’s hematoxylin, and mounted.


Statistical analysis
Statistical analysis was performed using PASW Statistics version 18 software. Results are expressed as mean±SD, unless indicated otherwise. Differences in the density of mast cells were compared between groups that were categorized according to disease parameters. The Kruskal-Wallis nonparametric test was used for analysis of more than two groups, and the Mann-Whitney U test was used for analysis of two groups. Spearman’s rank correlation coefficient was calculated to assess the correlation between the density of mast cells detected by toluidine blue and that by c-Kit. A P value less than 0.05 denoted the presence of a statistically significant difference.