Author/Editor     Kralj, Jana
Title     Določanje antigenov CD62P in CD63 s pretočnim citometrom za oceno aktiviranih trombocitov
Translated title     Detection of activated platelets by flow cytometric analysis of antigens CD62P and CD63
Type     članek
Source     Zdrav Vestn
Vol. and No.     Letnik 67, št. Suppl 1
Publication year     1998
Volume     str. I-57-9
Language     slo
Abstract     Background. Plateles may become activated in a number of disorders such as atherosclerosis, thrombosis and cancer. Therefore, the detection of activated platelets might facilitate the identification of certain thrombotic disorders. Previous methods for assaing platelet activity have been indirect, and quantitation has been difficult, precluding their use in the clinical area. Flow cytometry is a promising new method, using monoclonal antibodies specific for a glycoprotein markers of platelet activation expresssed on the membrane surface following activation. Methods. Cell surface expression of granule membrane antigens P-selectin (CD62P) and lysosome antigen CD63 were examined by flow cytometry in 30 healthy donors. We studied the conditions to prevent artifactual induction of platelet activation. Results. The proportion of platelets expressing antigens CD62P and CD63 in blood samples with paraformaldehyde was lower (<5%) than in samples with theophyillin (< 27%). The concentration-effect relationship were determined for the ADP, adrenaline, thrombin and collagen induced expression of CD62P and CD63. Conclusions. The results suggest that 1% solution of paraformaldehyde should be used for preventing of artifactual induction of platelet activation in blood samples. Platelets are likely to become activated by different agonist in different clinical situations. Therefore, it seems necessary to detect of activated platelets by a panel of monoclonal antibodies.
Summary     Izhodišča. Aktivne trombocite so ugotovili pri bolnikih z aterosklerozo, vensko trombozo in malignimi boleznimi. Določanje aktiviranih trombocitov je pomembno za odkrivanje tveganja za nastanek tromboze. Večina načinov za oceno trombocitne funkcije je posrednih. Nanje vplivajo številni dejavniki, ki omejujejo klinično uporabnost. pretočni citometer nam ponuja določanje aktiviranih trombocitov z uporabo monoklonskih protiteles. Kljub številnim prednostim, pa se pretočna citometrija za določanje aktiviranih trombocitov v klinični praksi še ni uveljavila. Preiskovanci in metode. Aktivirane trombocite smo določili pri 30 zdravih preiskovancih s protitelesi za antigena DC26P in CD63 pred in po spodbuditvi z ADP, ADP in ADR, trombinom in kolagenom. Preverili smo pogoje, ki preprečijo aktivacijo trombocitov med delom in zagotovijo ponovljivost izsledkov. Rezultati. V vzorcih krvi s paraformaldehidom smo ugotovili znatno manjši delež trombocitov z antigeni CD62P in CD 63 (< 5%) kot, v vzorcih krvi s teofilinom (< 27%). Ugotovili smo, da so koncentracije ADP 10 qmol/L, ADP in ADR 20 qmol/L, 0,17 E/mL trombina in 88 qg/mL kolagena zadostne da spodbudijo trombocite k premestitvi antigenov CD62P in CD63 na membrano, ne povzročijo pa medsebojnega zlepljanja. Določili smo referenčne vrednosti pred spodbuditvijo z izbranimi spodbujevalci in po njej. Zaključki. Menimo, da je za preprečevanje aktivacije trombocitov med določanjem antigenov CD62P in CD63 najbolj primerna uporaba 1% raztopine paraformaldehida. Deleži trombocitov z antigeni CD62P in CD63 se razlikujejo glede na izbrane spodbujevalce. Menimo, da je za celovito oceno aktiviranih trombocitov smiselno uporabiti več monoklonskih protiteles.
Descriptors     PLATELET ACTIVATION
P-SELECTIN
PLATELET MEMBRANE GLYCOPROTEINS
FLOW CYTOMETRY
ANTIBODIES, MONOCLONAL
ADENOSINE DIPHOSPHATE
THROMBIN
COLLAGEN