vestimИзвестия Национальной академии наук Беларуси. Серия медицинских наукProceedings of the National Academy of Sciences of Belarus, Medical series1814-60232524-2350The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"vestim-331Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEОПТИМИЗАЦИЯ АЛГОРИТМА ГЕЙТИРОВАНИЯ БАЗОФИЛОВ НА ПРОТОЧНОМ ЦИТОМЕТРЕ: МНОГОЦВЕТНЫЙ АНАЛИЗOPTIMIZATION OF BASOPHIL GATING ALGORITHM ON FLOW CYTOMETRY: MULTICOLOR ANALYSISРомановаИ. В.RamanavaI. U.

науч. сотрудник лаборатории иммунологии и клеточной биотехнологии. Республиканский научно-практический центр эпидемиологии и микробиологии

Researcher of the Laboratory for Immunology and Cellular Biotechnology

romanovairavlad@gmail.comГончаровА. Е.HancharouA. Y.

канд. мед. наук, зав. лабораторией иммунологии и клеточной биотехнологии. Республиканский научно-практический центр эпидемиологии и микробиологии

andrei.hancharou@gmail.comДудареваН. И.DudaravaN. I.

канд. мед. наук, зав. отделением аллергологии и профпатологии

Ph. D. (Med.), Head of the Depart-ment of Allergology and Occupational Pathology

lpu10gkb@rambler.ruРеспубликанский научно-практический центр эпидемиологии и микробиологииRepublican Scientific and Practical Center for Epidemiology and Microbiology10-я городская клиническая больница10th City Clinical Hospital201613012017041524Copyright © Романова И.В., Гончаров А.Е., Дударева Н.И., 20172017Романова И.В., Гончаров А.Е., Дударева Н.И.Ramanava I.U., Hancharou A.Y., Dudarava N.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestimed.belnauka.by/jour/article/view/331

Проанализировано 7 способов гейтирования базофилов с использованием многоцветного анализа на проточном цитометре. Показано, что наиболее эффективными и надежными способами гейтирования для проведения теста активации базофилов являются IgEhi и CD123+HLA-DR–. Проведены исследования стабильности экспрессии маркеров идентификации базофилов в зависимости от статуса активации базофилов. Установлено, что молекулы IgE и CD123, в отличие от молекул CD203c и CD193, не имеют значимых различий в интенсивности экспрессии при аллергенспецифической активации базофилов. Количество примеси других популяций в регионе базофилов при гейтировании с использованием маркеров IgEhi и  CD123+HLA-DR– минимально, что указывает на то, что данные маркеры идентификации могут быть с успехом использованы для проведения теста активации базофилов.

Seven strategies of basophil gating were analyzed using multicolor flow cytometry. It was shown that the most effective and reliable gating strategies for BAT were IgEhi and CD123+HLA-DR–. The stability of basophil identification markers depending on the activation status of basophils was assayed. It was established that IgE and CD123 molecules exhibited no significant difference unlike molecules CD203c and CD193. It was shown that the IgEhi and CD123+HLA-DR– regions had the lowest contamination by other cell subsets, which indicates that the both gating strategies may be used successfully for basophil activation test.

базофилыгейтированиепроточная цитометриятест активации базофиловbasophilsgating strategyflow cytometrybasophil activation testReferencesPros and cons of clinical basophil testing (BAT) / H. J. Hoffmann [et al.] // Curr. Allergy Asthma Rep. – 2016. – Vol. 16, N 8. – P. 56–68.Pros and cons of clinical basophil testing (BAT) / H. J. Hoffmann [et al.] // Curr. Allergy Asthma Rep. – 2016. – Vol. 16, N 8. – P. 56–68.The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease / H. J. Hoffmann [et al.] // Allergy. – 2015. – Vol. 70 – P. 1393–1405.The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease / H. J. Hoffmann [et al.] // Allergy. – 2015. – Vol. 70 – P. 1393–1405.Macglashan, D. Jr. / IgE and FcεRI regulation / D. Jr. Macglashan // Ann. N. Y. Acad. Sci. – 2005. – Vol. 1050. – P. 73–88.Macglashan, D. Jr. / IgE and FcεRI regulation / D. Jr. Macglashan // Ann. N. Y. Acad. Sci. – 2005. – Vol. 1050. – P. 73–88.Flowcytometric analysis of basophil counts in human blood and inaccuracy of hematology analyzers / S. Ducrest [et al.] // Allergy. – 2005. – Vol. 60. – P. 1446–1450.Flowcytometric analysis of basophil counts in human blood and inaccuracy of hematology analyzers / S. Ducrest [et al.] // Allergy. – 2005. – Vol. 60. – P. 1446–1450.Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2 / H. Hirai [et al.] // J. Exp. Med. – 2001. – Vol. 193, N 2. – P. 255–261.Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2 / H. Hirai [et al.] // J. Exp. Med. – 2001. – Vol. 193, N 2. – P. 255–261.Allergen-specific basophil suppression associated with clinical tolerance in patients with milk allergy / N. Wanich [et al.] // J. Allergy. Clin. Immunol. – 2009. – Vol. 123, N 4. – P. 789–794.Allergen-specific basophil suppression associated with clinical tolerance in patients with milk allergy / N. Wanich [et al.] // J. Allergy. Clin. Immunol. – 2009. – Vol. 123, N 4. – P. 789–794.The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors / H. J. Bühring [et al.] // Blood. – 1999. – Vol. 94, N 7. – P. 2343–2356.The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors / H. J. Bühring [et al.] // Blood. – 1999. – Vol. 94, N 7. – P. 2343–2356.Sainte-Laudy, J. Analysis of membrane expression of the CD63 human basophil activation marker. Applications to allergologic diagnosis / J. Sainte-Laudy, C. Vallon, J. C. Guérin // Allerg. Immunol. (Paris). – 1994. – Vol. 26, N 6. – P. 211–214.Sainte-Laudy, J. Analysis of membrane expression of the CD63 human basophil activation marker. Applications to allergologic diagnosis / J. Sainte-Laudy, C. Vallon, J. C. Guérin // Allerg. Immunol. (Paris). – 1994. – Vol. 26, N 6. – P. 211–214.Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. technical issues / A. L. De Week [et al.] // J. Investig. Allergol. Clin. Immunol. – 2008. – Vol. 18, N 3. – P. 143–155.Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. technical issues / A. L. De Week [et al.] // J. Investig. Allergol. Clin. Immunol. – 2008. – Vol. 18, N 3. – P. 143–155.WEASEL for flow cytometry [Electronic resource] // The Walter and Eliza Hall Institute of Medical Research. – Mode of access: http://www.frankbattye.com.au/Weasel/index.html. – Date of access: 19.06.2016.WEASEL for flow cytometry [Electronic resource] // The Walter and Eliza Hall Institute of Medical Research. – Mode of access: http://www.frankbattye.com.au/Weasel/index.html. – Date of access: 19.06.2016.BD FACSDiva™ Software [Electronic resource]. – Mode of access: http://www.bdbiosciences.com/instruments/software/facsdiva/index.jsp. – Date of access: 19.06.2016.BD FACSDiva™ Software [Electronic resource]. – Mode of access: http://www.bdbiosciences.com/instruments/software/facsdiva/index.jsp. – Date of access: 19.06.2016.Реброва, О. Ю. Статистический анализ медицинских данных. Применение пакета прикладных программ Statistica / О. Ю. Реброва. – М., 2008. – 312 с.Реброва,   О. Ю. Статистический анализ медицинских данных. Применение пакета прикладных программ Statistica / О. Ю. Реброва. – М., 2008. – 312  с.A guide to modern statistical analysis of immunological data [Electronic resource] / B. Genser [et al.] // BMC Immunol. – 2007. – Vol. 8, N 27. – Mode of access: http://www.biomedcentral.com/1471-2172/8/27. – Date of access: 19.06.2016.A guide to modern statistical analysis of immunological data [Electronic resource] / B. Genser [et al.] // BMC Immunol. – 2007. – Vol. 8, N 27. – Mode of access: http://www.biomedcentral.com/1471-2172/8/27. – Date of access: 19.06.2016.CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation /E. M. Sturm [et al.] // Cytometry. Part B (Clin. Cytometry). – 2010. – Vol. 78B. – P. 308–318.CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation /E. M. Sturm [et al.] // Cytometry. Part B (Clin. Cytometry). – 2010. – Vol. 78B. – P. 308–318.Recombinant allergens promote expression of CD203c on basophils in sensitized individuals / A. W Hauswirth [et al.] // J. Allergy Clin. Immunol. – 2002. – Vol. 110, N 1. – P. 102–109.Recombinant allergens promote expression of CD203c on basophils in sensitized individuals / A. W Hauswirth [et al.] // J. Allergy Clin. Immunol. – 2002. – Vol. 110, N 1. – P. 102–109.Macglashan, D. Jr. Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes individuals / D. Jr. Macglashan // Clin. Exp. Allergy. – 2010. – Vol. 40, N 9. – P. 1365–1377.Macglashan, D. Jr. Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes individuals / D. Jr. Macglashan // Clin. Exp. Allergy. – 2010. – Vol. 40, N 9. – P. 1365–1377.Bühring, H. J. The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis / H. J. Bühring, A. Streble, P. Valent // Int. Arch. Allergy Immunol. – 2004. – Vol. 133, N 4. – P. 317–329.Bühring, H. J. The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis / H. J. Bühring, A. Streble, P. Valent // Int. Arch. Allergy Immunol. – 2004. – Vol. 133, N 4. – P. 317–329.Selecting Reagents for Multicolor Flow Cytometry [Electronic resource] / H. Maecker, J. Trotter // BD Biosciences Application Note. – 2012. – Vol. 6. – Mode of access: web.mit.edu/flowcytometry/www/Becton%20Dickinson%20Multicolor_AppNote.pdf. – Date of access: 19.06.2016.Selecting Reagents for Multicolor Flow Cytometry [Electronic resource] / H. Maecker, J. Trotter // BD Biosciences Application Note. – 2012. – Vol. 6. – Mode of access: web.mit.edu/flowcytometry/www/Becton%20Dickinson%20Multicolor_AppNote.pdf. – Date of access: 19.06.2016.Comparison between antibodies from different providers: determination of median fluorescent intensities and stain indices [Electronic resource] / K. Reynvoet [et al.] // MACS&more. – 2012. – Vol. 14. – Mode of access: miltenyibiotec.com/~/media/Images/Products/Import/0010000/IM0010013.ashx. – Date of access: 19.06.2016.Comparison between antibodies from different providers: determination of median fluorescent intensities and stain indices [Electronic resource] / K. Reynvoet [et al.] // MACS&more. – 2012. – Vol. 14. – Mode of access: miltenyibiotec.com/~/media/Images/Products/Import/0010000/IM0010013.ashx. – Date of access: 19.06.2016.The evolution of guidelines for the validation of flow cytometric methods / L. Du [et al.] // Int. Jnl. Lab. Hem. – 2015. – Vol. 37. – Suppl. 1. – Р. 3–10.The evolution of guidelines for the validation of flow cytometric methods / L. Du [et al.] // Int. Jnl. Lab. Hem. – 2015. – Vol. 37. – Suppl. 1. – Р. 3–10.Chirumbolo, S. CCR3 as a Single Selection Marker Compared to CD123/HLADR to Isolate Basophils in Flow Cytometry: Some Comments / S. Chirumbolo, R. Ortolani, A. Vella // Cytometry A. – 2011. – Vol. 79, N 2. – Р. 102–106.Chirumbolo, S. CCR3 as a Single Selection Marker Compared to CD123/HLADR to Isolate Basophils in Flow Cytometry: Some Comments / S. Chirumbolo, R. Ortolani, A. Vella // Cytometry A. – 2011. – Vol. 79, N 2. – Р. 102–106.Sturm, E. M., Kranzelbinder, B., Heinemann, A., Groselj-Strele, A., Aberer, W. and Sturm, G. J. (2010) “CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation”, Cytometry Part B (Clinical Cytometry), vol. 78B, pp. 308-318.Sturm, E. M., Kranzelbinder, B., Heinemann, A., Groselj-Strele, A., Aberer, W. and Sturm, G. J. (2010) “CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation”, Cytometry Part B (Clinical Cytometry), vol. 78B, pp. 308-318.Hauswirth, A. W., Natter, S., Ghannadan, M., Majlesi, Y., Schernthaner, G. H., Sperr, W. R., Bührving, H. J., Valenta, R. and Valent, P. (2002) “Recombinant allergens promote expression of CD203c on basophils in sensitized individuals”, Journal of Allergy and Clinical Immunology, vol. 110, no. 1, pp. 102-109.Hauswirth, A. W., Natter, S., Ghannadan, M., Majlesi, Y., Schernthaner, G. H., Sperr, W. R., Bührving, H. J., Valenta, R. and Valent, P. (2002) “Recombinant allergens promote expression of CD203c on basophils in sensitized individuals”, Journal of Allergy and Clinical Immunology, vol. 110, no. 1, pp. 102-109.Macglashan, D. Jr. (2010) “Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes individuals”, Clinical and experimental allergy, vol. 40, no. 9, pp. 1365-1377.Macglashan, D. Jr. (2010) “Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes individuals”, Clinical and experimental allergy, vol. 40, no. 9, pp. 1365-1377.Bühring, H. J., Streble, A. and Valent, P. (2004) “The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis”, International Archives of Allergy and Immunology, vol. 133, no. 4, pp. 317-329.Bühring, H. J., Streble, A. and Valent, P. (2004) “The basophil-specific ectoenzyme E-NPP3 (CD203c) as a marker for cell activation and allergy diagnosis”, International Archives of Allergy and Immunology, vol. 133, no. 4, pp. 317-329.Maecker, H. and Trotter, J. (2012) “Selecting Reagents for Multicolor Flow Cytometry. BD Biosciences”, Application Note, vol. 6. Available at: web.mit.edu/flowcytometry/www/Becton%20Dickinson%20Multicolor_AppNote.pdf (Accessed 19 June 2016).Maecker, H. and Trotter, J. (2012) “Selecting Reagents for Multicolor Flow Cytometry. BD Biosciences”, Application Note, vol. 6. Available at: web.mit.edu/flowcytometry/www/Becton%20Dickinson%20Multicolor_AppNote.pdf (Accessed 19 June 2016).Reynvoet, K., Vermaut, S., de Colvenaer, V. and Frings P. (2012) “Comparison between antibodies from different providers: determination of median fluorescent intensities and and stain indices”, MACS&more, vol. 14. Available at: miltenyibiotec. com/~/media/Images/Products/Import/0010000/IM0010013.ashx (Accessed 19 June 2016).Reynvoet, K., Vermaut, S., de Colvenaer, V. and Frings P. (2012) “Comparison between antibodies from different providers: determination of median fluorescent intensities and and stain indices”, MACS&more, vol. 14. Available at: miltenyibiotec. com/~/media/Images/Products/Import/0010000/IM0010013.ashx (Accessed 19 June 2016).Du, L., Grover, A., Ramanan, S. and Litwinv, V. (2015) “The evolution of guidelines for the validation of flow cytometric methods”, International Journal of Laboratory Hematology, vol. 37, suppl. 1, pp. 3-10.Du, L., Grover, A., Ramanan, S. and Litwinv, V. (2015) “The evolution of guidelines for the validation of flow cytometric methods”, International Journal of Laboratory Hematology, vol. 37, suppl. 1, pp. 3-10.Chirumbolo S., Ortolani R. and Vella, A. (2011) “CCR3 as a Single Selection Marker Compared to CD123/HLADR to Isolate Basophils in Flow Cytometry: Some Comments”, Cytometry A, vol. 79, no. 2, pp. 102-106.Chirumbolo S., Ortolani R. and Vella, A. (2011) “CCR3 as a Single Selection Marker Compared to CD123/HLADR to Isolate Basophils in Flow Cytometry: Some Comments”, Cytometry A, vol. 79, no. 2, pp. 102-106.

The authors declare that there are no conflicts of interest present.