rss
Thorax 1999;54:124-130 doi:10.1136/thx.54.2.124
  • Original article

Objective quantitative analysis of eosinophils and bronchial epithelial cells in induced sputum by laser scanning cytometry

  1. G Woltmanna,
  2. R J Warda,
  3. F A Symona,
  4. D A Rewb,
  5. I D Pavorda,
  6. A J Wardlawa
  1. aDivision of Respiratory Medicine, bDepartment of Surgery, cUniversity of Leicester, Glenfield Hospital, Groby Road, Leicester LE3 9PQ, UK
  1. Dr AJ Wardlaw.
  • Received 24 April 1998
  • Revision requested 17 July 1998
  • Revised 17 September 1998
  • Accepted 7 October 1998

Abstract

BACKGROUND Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide.

METHODS LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using α-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-α-human-MBP monoclonal antibody or mouse-α-human-cytokeratin antibody and goat-α-mouse Oregon Green conjugated second antibody.

RESULTS Sputum induction provided a mean (SE) of 0.99 (0.2) × 106 cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation.

CONCLUSION Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.

Footnotes

    This Article

    1. Abstract
    2. Full text
    3. PDF

    Register for free content

    The full back archive is now available for all BMJ Journals. Institutional subscribers may access the entire archive as part of their subscription. Personal subscribers will also have access to all content when logged in. Non-subscribers who register have free access to all articles published before 2006 right back to volume 1 issue 1. Register here to access the free archive of all BMJ Journals.

    Don't forget to sign up for content alerts so you keep up to date with all the articles as they are published.