Novel approach for discrimination of eosinophilic granulocytes and evaluation of their surface receptors in a multicolor fluorescent histological assessment

Abstract

Eosinophilic granulocytes mediate immune responses against multicellular parasites and are also the main contributor to such pathological conditions as allergy and asthma. Eosinophils are discriminated with eosin staining in conventional histology using light microscopy. However, molecular detection of antigens and the widely introduced automated analyzers usually require fluorescent markers to allow quantitative determination. Surprisingly, there is no selective CD marker to differentiate eosinophils and basophils. Recently reported analogs to replace hematoxylin and eosin staining for immune-histochemical applications such as DRAQ5 & eosin are also unsuitable due to wide fluorescent spectra. Different combinations of fluorescent dyes were tested using fluorescent microscopy aimed to develop a simple and specific method for detecting eosinophilic granules, DNA and surface receptors, the approach was used for evaluation of IgE levels (total and specific to casein) on cells of patients suffering from cow milk allergy. We were able to achieve selective visualization of eosinophil granules using aniline blue dye by modifying the method of Berretty & Cormane (1978) and detecting emission at 440nm; this allowed simultaneous staining of blood smears with anti-IgE-FITC (emission at 520 nm) and casein-FITC, detection of DNA with propidium iodide (em. 590 nm), and also provided specific metachromatic signal of eosinophils in the NIR region (em. ~700 nm) with subsequent quantification of fluorescent signal. Application of this approach to clinical cases revealed increased IgE levels and increased casein-binding targets on eosinophils in 3 patients with cow milk allergy compared to 2 healthy donors, demonstrating the general usefulness of the approach.

Keywords

• allergy
• clinical case
• detection
• eosinophilic granulocytes
• histology
• ige
• milk