title: 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.
  
  
  CONCLUSIONS
  The digital procedure proved accurate compared to the reference standard, producing reliable results for whole tissue sections.
  
  
  BACKGROUND
  ClinicalTrials.gov [ NCT00740948 ]. Registered 22 August 2008.
authors.affiliation: Pathology Department, Brest University Hospital, Brest, France. sebastian.costa@chu-brest.fr.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Costa
authors.affiliation: Biology Department, Brest University Hospital, Brest, France. sacha@labsquare.org.
authors.forename: Sacha
authors.initials: S
authors.lastname: Schutz
authors.affiliation: Rheumatology Department, Brest University Hospital, Brest, France. divi.cornec@chu-brest.fr.
authors.forename: Divi
authors.initials: D
authors.lastname: Cornec
authors.affiliation: Pathology Department, Brest University Hospital, Brest, France. arnaud.uguen@chu-brest.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Uguen
authors.affiliation: Pathology Department, Brest University Hospital, Brest, France. isabelle.quintin-roue@chu-brest.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Quintin-Roué
authors.affiliation: Pathology Department, Vannes General Hospital, Vannes, France. agnes.lesourd@ch-bretagne-atlantique.fr.
authors.forename: Agnès
authors.initials: A
authors.lastname: Lesourd
authors.affiliation: Rheumatology Department, Hôtel Dieu University Hospital, Nantes, France. jeanmarie.berthelot@chu-nantes.fr.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Berthelot
authors.affiliation: Department of Internal Medicine, Nord-de-France University, Claude-Huriez Hospital, Lille, France. eric.hachulla@chru-lille.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Hachulla
authors.affiliation: Department of Internal Medicine, Nord-de-France University, Claude-Huriez Hospital, Lille, France. pierre-yves.hatron@chru-lille.fr.
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Hatron
authors.affiliation: Rheumatology Department, Amiens University Hospital, Amiens, France. Goeb.vincent@chu-amiens.fr.
authors.forename: Vincent
authors.initials: V
authors.lastname: Goeb
authors.affiliation: Rheumatology Department, Rouen University Hospital, Rouen, France. olivier.vittecoq@chu-rouen.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Vittecoq
authors.affiliation: EA 2216-ESPRI 29, Brest University, Brest, France. pers@univ-brest.fr.
authors.forename: Jacques Olivier
authors.initials: JO
authors.lastname: Pers
authors.affiliation: Pathology Department and EA 4685, Brest University Hospital, Brest, France. pascale.marcorelles@chu-brest.fr.
authors.forename: Pascale
authors.initials: P
authors.lastname: Marcorelles
authors.affiliation: Rheumatology Department and EA 2216-ESPRI 29, Brest University Hospital, Brest, France. alain.saraux@chu-brest.fr.
authors.forename: Alain
authors.initials: A
authors.lastname: Saraux
authors.affiliation: Rheumatology Department and EA 2216-ESPRI 29, Brest University Hospital, Brest, France. valerie.devauchelle-pensec@chu-brest.fr.
authors.forename: Valérie
authors.initials: V
authors.lastname: Devauchelle-Pensec
mesh_terms: D000328:Adult; D000368:Aged; D001402:B-Lymphocytes / Q000473:pathology*; D005260:Female; D006801:Humans; D018655:Lymphocyte Count / Q000592:standards* / Q000639:trends; D008297:Male; D008875:Middle Aged; D012470:Salivary Glands, Minor / Q000473:pathology*; D012859:Sjogren's Syndrome / Q000473:pathology*; D013601:T-Lymphocytes / Q000473:pathology*