Ukr.Biochem.J. 2020; Volume 92, Issue 2, Mar-Apr, pp. 99-106


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

G. Bila1, M. Schneider1, S. Peshkova1, B. Krajnik2, L. Besh3, A. Lutsyk1, O. Matsyura3*, R. Bilyy1*

1Department of Histology, Cytology and Embryology, Danylo Halytsky Lviv National Medical University, Ukraine;
2Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Poland;
3Department of Pediatrics 2, Danylo Halytsky Lviv National Medical University, Ukraine;

Received: 09 January 2020; Accepted: 27 March 2020

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: , , , , , ,


  1. Ramirez GA, Yacoub MR, Ripa M, Mannina D , Cariddi A , Saporiti N, Ciceri F, Castagna A, Colombo G, Dagna L. Eosinophils From Physiology to Disease: A Comprehensive Review. Biomed Res Int.  2018;2018:9095275. PubMed, PubMedCentral, CrossRef
  2. Boeltz S, Amini P, Anders HJ, Andrade F, Bilyy R, Chatfield S, Cichon I, Clancy DM, Desai J, Dumych T, Dwivedi N, Gordon RA, Hahn J, Hidalgo A, Hoffmann MH, Kaplan MJ, Knight JS, Kolaczkowska E, Kubes P, Leppkes M, Manfredi AA, Martin SJ, Maueröder C, Maugeri N, Mitroulis I, Munoz LE, Nakazawa D, Neeli I, Nizet V, Pieterse E, Radic MZ, Reinwald C, Ritis K, Rovere-Querini P, Santocki M, Schauer C, Schett G, Shlomchik MJ, Simon HU, Skendros P, Stojkov D, Vandenabeele P, Berghe TV, van der Vlag J, Vitkov L, von Köckritz-Blickwede M, Yousefi S, Zarbock A, Herrmann M. To NET or Not to NET:current Opinions and State of the Science Regarding the Formation of Neutrophil Extracellular Traps. Cell Death Differ. 2019;26(3):395-408. PubMed, PubMedCentral, CrossRef
  3. Mukherjee M, Lacy P, Ueki S. Eosinophil Extracellular Traps and Inflammatory Pathologies-Untangling the Web! Front Immunol. 201;9:2763. PubMed, PubMedCentral, CrossRef
  4. Paryzhak SYa, Dumych TI, Bila GI, Bilyy RO. Application of air pouch model for evaluation the ability of immune cells to sequester foreign nano- and microparticles. Exp Clin Physiol Biochem. 2019;2019(1): 52-57. CrossRef
  5. Bila G, Peshkova S, Dumych T, Bilyy R. Natural cholesterol nanocrystals in gall material and their interaction with neutrophilic granulocytes. In: PhoBiA Annual Nanophotonics International Conference. Wroclaw, 2019. p. 62.
  6. ThermoFisher. Immune cell guide: Antigens, human and mouse. 2017. 84 p.
  7. Elfer KN, Sholl AB, Wang M, Tulman DB, Mandava SH, Lee BR, Brown JQ. DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues. PLoS One. 2016;11(10):e0165530. PubMed, PubMedCentral, CrossRef
  8. Wollenberg A, Barbarot S, Bieber T, Christen-Zaech S, Deleuran M, Fink-Wagner A, Gieler U, Girolomoni G, Lau S, Muraro A, Czarnecka-Operacz M, Schäfer T, Schmid-Grendelmeier P, Simon D, Szalai  Z, Szepietowski JC, Taïeb A, Torrelo A , Werfel T , Ring J.  Consensus-based European Guidelines for Treatment of Atopic Eczema (Atopic Dermatitis) in Adults and Children: Part I. J Eur Acad Dermatol Venereol. 2018;32(5):657-682. PubMed, CrossRef
  9. Berretty PJ, Cormane RH. The Aniline Blue Fluorescence Staining of Eosinophilic Granulocytes. Br J Dermatol. 1978;99(4):377-382. PubMed, CrossRef
  10. Hermanson GT. Bioconjugate Techniques [Internet]. 3rd ed. London: Academic Press, 2013. 1200 p.
  11. Bilyy R, Tomyn A, Kit Y, Podhorodecki A, Misiewicz J, Nyk M, Strek W, Stoika R. Detection of dying cells using lectin-conjugated fluorescent and luminescent nanoparticles. Materialwiss Werkstofftech. 2009; 40(4): 234-237. CrossRef
  12. Sojka B, Podhorodecki A, Banski M, Misiewicz J, Drobczynski S, Dumych T, Lutsyk MM, Lutsyk A, Bilyy R. β-NaGdF4:Eu3+ nanocrystal markers for melanoma tumor imaging. RSC Adv. 2016; 6(63): 57854–57862. CrossRef
  13. Wal JM. Cow’s milk allergens. Allergy. 1998; 53(11): 1013–1022. PubMed, CrossRef
  14. Wal JM. Cow’s milk proteins/allergens. Ann Allergy Asthma Immunol. 2002; 89(6 Suppl 1): 3-10. PubMed, CrossRef
  15. Grama S, Boiko N, Bilyy R, Klyuchivska O, Antonyuk V, Stoika R, Horak D. Novel fluorescent poly(glycidyl methacrylate) – silica microspheres. Eur Polym J. 2014; 56: 92-104.   CrossRef

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