Identification of circulating endothelial cells and endothelial progenitor cells by flow cytometry

Endotelijske cirkulirajoče in endotelijske prekurzorske celice predstavljajo obetavne označevalce endotelijske poškodbe oziroma regeneracijske sposobnosti žilja, vendar so omenjene celice v krvi zelo redke, kar otežuje njihovo določanje. Najpogostejši metodi za njihovo določanje sta imunomagnetna izolacija in pretočna citometrija. Slednja omogoča analizo velikega števila celic v relativno kratkem času in možnost uporabe kombinacije različnih celičnih označevalcev. To je še posebej pomembno, saj nimamo označevalca, ki bi bil popolnoma specifičen za te celice. Z namenom prepoznave cirkulirajočih in prekurzorskih endotelijskih celic v periferni krvi s pretočno citometrijo smo razvili protokol za njihovo določitev. Njihovo število smo določili s pomočjo dvostopenjske metode, nespecifično vezavo pa smo za vsak vzorec ocenili z uporabo izotipske kontrole. Cirkulirajoče endotelijske celice smo prepoznali kot žive celice z jedrom, ki so CD34 pozitivne, CD45 negativne ali šibko pozitivne, CD133 negativne in CD146 pozitivne. Endotelijske prekurzorske pa so žive celice z jedrom, ki so CD34 pozitivne, CD45 šibko pozitivne, CD133 in CD309 pozitivne. Dokazali smo, da je metoda ponovljiva in da število krvotvornih matičnih celic korelira s številom, določenim s standardnim postopkom za njihovo določanje. Analiza vzorcev mora biti zaradi slabe stabilnosti celic izvedena čim prej po odvzemu vzorcev. Točnost smo dokazali z analizo vzorcev bolnikov s sladkorno boleznijo tipa 1. Ugotovili smo, da se je število cirkulirajočih endotelijskih celic značilno zmanjšalo pri tistih bolnikih, ki so 3 mesece prejemali zdravilo semaglutid. Število endotelijskih prekurzorskih celic pa se je statistično značilno povišalo pri bolnikih na 3-mesečni terapiji z zdravilom semaglutid ali empagliflozin. Medtem v kontrolni skupini med meritvami v obeh časovnih točkah nismo ugotovili statistično značilnih razlik. Ker pa so endotelijske prekurzorske celice v periferni krvi zelo redke, bi bilo treba ponovno oceniti ustreznost imunofenotipa za uporabo v vsakdanji klinični praksi.Circulating endothelial and endothelial progenitor cells are a promising marker of endothelial damage and endothelial regeneration potential. However, their quantification is difficult due to their rareness in the peripheral blood. The most commonly used methods for their identification are immunomagnetic separation and flow cytometry. An advantage of flow cytometry is that a large number of cells can be analysed in a relatively short time in addition to the fact that a combination of various cell markers can be used at the same time. This is important, because we do not have marker that is completely specific for circulating endothelial and endothelial progenitor cells. In order to identify these cells in the peripheral blood by flow cytometry, we developed a protocol for their determination, where an isotype control was used to eliminate nonspecific events. The absolute number was determined with double counting method. Circulating endothelial cells were identified as live nucleated cells that are CD34 positive, CD45 negative or dimly positive, CD133 negative and CD146 positive. Furthermore, endothelial progenitor cells were recognised as live nucleated cells, that are CD34 positive, CD45 dimly positive, CD133 and CD309 positive. We proved good repeatability of our method and good correlation with the standard technique for hematopoietic steam cells determination. Because of the poor stability of circulating endothelial and endothelial progenitor cells the analysis must be done as soon as possible after the venepuncture. Accuracy was proven with the analysis of blood samples from patients with type 1 diabetes. Wilcoxon test for paired samples showed, that the number of circulating endothelial cells decreased significantly in patients receiving semaglutide for 3 months. In addition, the number of endothelial progenitor cells increased significantly after 3 months therapy with semaglutide and empagliflozin. On the other hand, in the control group significant difference between the number of circulating endothelial and endothelial progenitor cells at both time points was not observed. Due to the rarity of endothelial progenitor cells in the peripheral blood, their immunophenotype should be reevaluated before implementation into clinical practice