Karen M. Britten

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from endoscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol methacrylate (GMA). GMA embedding generated an immunophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosinophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.

Airway Inflammation and Atopic Asthma: A Comparative Bronchoscopic Investigation

Flexible fibre-optic bronchoscopy under local anaesthesia has been used to investigate the cellular airway events in atopic asthma. The findings have been compared to those from atopic individuals without asthma and non-atopic healthy controls, in an attempt to discern those changes relevant to clinical disease expression. Immunohistochemical and electron-microscopic analyses of airway biopsies identified that an atopic diathesis is associated with tissue eosinophil infiltration and mast cell degranulation. The eosinophilia was greatest in those atopic individuals with asthma. Flow-cytometric analysis of airway lavage revealed significantly enhanced T lymphocyte activation in clinical asthma. These findings are consistent with the hypothesis that T lymphocyte activation, through cytokine release, amplifies the tissue eosinophilia in asthma and that this combination is associated with clinical disease expression.

Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques

SummaryThe fixation and drying regimes for frozen sections and cytocentrifuge preparations for the demonstration of surface antigens, such as immunoglobulins and iron binding proteins, vary enormously between different groups of workers. A method using freeze-dried sections and acetone fixation was compared with 16 other methods of fixation and found to be the best for tissue preservation and antigen demonstration. Freeze drying was found to improve the cytological preservation of air-dried sections considerably.

Effect of inhaled platelet-activating factor on bronchial inflammation in atopic non-asthmatic subjects

We have investigated the effect of inhaled platelet-activating factor (PAF) on bronchial mucosal inflammation in 6 atopic non-asthmatic subjects in a double-blind, placebo-controlled, randomised and crossover study. On 2 study periods at least 4 weeks apart, fiberoptic bronchoscopy was performed 24 h after inhalation of either 200 micrograms PAF or methacholine (control) to obtain endobronchial biopsies. Immunocytochemistry using antibodies for trypase (AA1) and eosinophil cationic protein (EG2) was performed to enumerate mast cells and eosinophils, respectively, in the bronchial submucosa. Median values of AA1+ cells and EG2+ cells did not differ significantly after inhalation of PAF or control (23.8 vs. 39 and 6 vs. 8/mm2, respectively, PAF vs. control, non-significant). Our findings suggest that within 24 h of inhaling a bronchoconstrictor dose of PAF, this agonist does not induce bronchial hyperresponsiveness or mucosal inflammation in atopic non-asthmatic subjects. However, because of the small number of subjects studied, these preliminary data should be interpreted with caution.