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Authors’ reply
  1. Y Qiu,
  2. P K Jeffery
  1. Lung Pathology, Department of Gene Therapy, Imperial College London, Royal Brompton Hospital, London, UK
  1. Dr P K Jeffery, Lung Pathology, Department of Gene Therapy, Imperial College London, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK; p.jeffery{at}imperial.ac.uk

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We thank Monteseirín and Vega for their comments concerning the specificity of the clone EG2 (Pharmacia & UpJohn Diagnostics AB, Uppsala, Sweden), mouse anti-human eosinophil cationic protein (ECP) monoclonal antibody used by us (Thorax 2007;62:475–82). We read with interest their results of isolated cells,1 which lead the authors to suggest caution in our interpretation of paraffin wax embedded bronchial biopsies from patients experiencing severe exacerbations of asthma. We respond respectfully and as follows.

Firstly, in our experience and those of the originator (Per Venge; recent personal communication), the cells invariably stained with EG2 have the characteristic morphology of eosinophils (ie, bi-lobed granular cells) whereas neutrophils stain rarely and too weakly to be included in our counts of formalin fixed paraffin wax embedded tissue. Secondly, while it is recognised that monoclonal antibodies are highly specific for the epitopes against which they have been raised, it is also widely appreciated that any particular epitope may be present on more than one cell type, or become expressed at different stages of cell maturation or activation. For example, dependent on the manufacturer and clone applied, the anti-CD56 monoclonal anti-human antibody (used by many to detect natural killer cells) may also detect a subset of CD4+ or CD8+ cells and even neurons, astrocytes and Schwann cells. Thus despite what manufacturers may claim, we have always been careful to conduct pilot studies in which we make comparisons against non-immunostains, in this case, of eosinophils (eg, chromotrope 2R), first titrating the antibodies that we plan to use and for each particular fixation and embedding protocol. Moreover, in order to reduce further non-specific staining due to any endogenous peroxidase present in large quantities in neutrophils and macrophages, we routinely apply the alkaline phosphatase anti-alkaline phosphatase technique2 rather than use a peroxidase based method.

Finally, in our study, we compared EG2 positive and neutrophil elastase (Ne) positive cells in bronchial biopsies between patients with stable asthma and asthmatics experiencing severe exacerbations. We found, in the latter, that while there was a significant increase in Ne positive cells, the numbers of EG2 positive cells did not increase significantly. If the concerns expressed were to be realised, it would be expected that the increase in EG2 positive cells would have followed closely that of Ne positive cells, which was not the case.

In conclusion, while we appreciate that other markers (eg, anti-MBP or anti-EPO) may also be used to mark eosinophils, for formalin fixed paraffin wax embedded tissue, the continued application of EG2 allows us to compare our findings with those previously done by both ourselves and by others. We consider that application of EG2 antibody to detect eosinophils remains a robust and interpretable technique, particularly by those appropriately trained and experienced in quantitative histopathology. The novel findings kindly communicated by Monteseirín and Vega are interesting and important, but caution must also be exercised in extrapolating from the results of such studies on isolated cells to those of tissues fixed and embedded, a process known to alter protein tertiary structure and hence the expression/accessibility of the epitopes of interest. We trust that these findings and discussion are helpful in immunohistological studies of inflammatory conditions, both in the lung and in general.

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  • Competing interests: None.

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