B-cell and T-cell quantification in minor salivary glands in primary Sjögren's syndrome: development and validation of a pixel-based digital procedure

Abstract

Background: Evaluating lymphocytic infiltration of minor salivary gland biopsy in primary Sjögren's syndrome is challenging. We developed and evaluated a digital method for quantifying B and T lymphocytes in whole minor salivary gland biopsy slides.

Methods: Minor salivary gland biopsies were immunostained with anti-CD20/anti-CD3 antibodies using red/brown chromogens. Slides were digitised and spliced into mosaics of smaller JPEG format images in which red and brown pixels were counted. ImageJ Cell counter was used for validation. Agreement between the digital and manual methods was evaluated using Bland-Altman plots and the interclass correlation coefficient. External validation relied on the Chisholm-Mason, Tarpley, and focus-score methods.

Results: Of 62 minor salivary gland biopsy slides, 61.3 % had a Chisholm-Mason grade ≥ III or a focus score ≥1. The number of pixels correlated well with manual cell counts (r = 0.95 for red pixels vs. B cell count and r = 0.91 for brown pixels vs. T cell count). Interclass correlation coefficients between digital and manual counts were excellent (0.92 for B/T cells). B-cell proportion showed a significant positive correlation with the focus score (Spearman's coefficient 0.463, p < 0.0001). Median B-cell proportion was lower in minor salivary gland biopsies with Chisholm grades I-II (2.5 % (0.2-13.9)) than III-IV (30.0 % (15.5-45.2)) and increased with Tarpley's class (1, 2.2 % (0.2-6.6); 2, 27.2 % (13.0-38.9); and 3-4, 48.5 % (29.4-56.4); p < 0.001 for all comparisons). Minor salivary gland biopsy B-cell proportion was also significantly correlated with several markers of clinical and biological activity of the disease, especially with markers of systemic B-cell hyperactivation.

Conclusion: The digital procedure proved accurate compared to the reference standard, producing reliable results for whole tissue sections.

Trial registration: ClinicalTrials.gov [ NCT00740948 ]. Registered 22 August 2008.

Figures

Fig. 1

The digital pixel counting procedure. a A minor salivary gland biopsy section on a standard glass slide was scanned using the NanoZoomer HT Scan system. b The NDPI file thus obtained was divided into several JPEG format files, which restored the original image if reassembled. The JPEG format files were managed by the algorythm (source code, available on https://github.com/dridk/bgsa-ndpi). c The signal in a given pixel was partitioned using a linear unmixing algorithm with simple red/green/blue imagery

Fig. 2

Micrographs of four JPEG format images showing the manual-count mask used by the ImageJ Cell counter. Stained B cells (in red) are marked with a dark blue dot and stained T cells (in brown) with a light blue dot. a First JPEG format image. Manual B-/T-cell counts: 135/361 (investigator 1) and 159/406 (investigator 2). Digital B-/T-cell count: 120/423. b Second JPEG format image. Manual B-/T-cell counts: 689/1067 (investigator 1) and 751/1089 (investigator 2). Digital B-/T-cell count: 532/1014. c Third JPEG-format image. Manual B-/T-cell count: 41/356 (investigator 1) and 50/426 (investigator 2). Digital B-/T-cell count: 36/410. d Fourth JPEG format image. Manual B-/T-cell counts: 469/860 (investigator 1) and 448/841 (investigator 2). Digital B-/T-cell count: 599/820

Fig. 3

Comparison of manual and digital counts. a Relations between the numbers of red and brown pixels and the manual counts of B and T cells, respectively. b Bland-Altman plots of digital versus manual counts for 31 JPEG format images. There is no tendency to under- or overestimate counts lower than 200 cells. With counts greater than 200 cells, our in-house software slightly overestimated the manual counts. SD Standard deviation

Fig. 4

Association between proportion of B cells and infiltrate severity. The proportion of B cells (computed as B-cell count/(B-cell + T-cell count)) was related to four markers of infiltrate severity: Chisholm-Mason grade (a), focus score (b), presence of germinal centres (c), and Tarpley’s class (d). The proportion of B cells varied significantly with the values of these severity markers (p < 0.0001)

Fig. 5

Correlations linking proportion of B cells to clinical and laboratory markers for disease activity. The proportion of B cells within minor salivary glands correlated with the intensity of oral dryness (visual analogue scale (VAS) score) (a), systemic activity (Eular Sjögren’s Syndrome Disease Activity Index; ESSDAI) (b), and biological markers for B-cell activity (serum IgG, anti-SSA antibody, and free light chains levels) (d, e, f), but not with unstimulated whole salivary flow (UWSF) (c)