{"id":2239,"date":"2021-09-19T22:50:23","date_gmt":"2021-09-19T20:50:23","guid":{"rendered":"https:\/\/www.newslab.sk\/porovnanie-analyzy-dostickovych-glykoproteinov-v-celej-krvi-a-v-plazme-bohatej-na-dosticky\/"},"modified":"2021-09-20T07:00:00","modified_gmt":"2021-09-20T05:00:00","slug":"porovnanie-analyzy-dostickovych-glykoproteinov-v-celej-krvi-a-v-plazme-bohatej-na-dosticky","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/porovnanie-analyzy-dostickovych-glykoproteinov-v-celej-krvi-a-v-plazme-bohatej-na-dosticky\/","title":{"rendered":"Comparison of platelet glycoprotein analysis in the wholeh blood and plasma rich in platelets"},"content":{"rendered":"

*A rare case of autochthonous human dirofilariasis with the manifestation of pseudotumor of the epididymis caused by helminth Dirofilaria repens<\/strong><\/span><\/p>\n

 <\/p>\n

Krvn\u00e9 do\u0161ti\u010dky s\u00fa bezjadrov\u00e9 \u00falomky cytoplazmy vznikaj\u00face z megakaryocytov v kostnej dreni. Denn\u00e1 produkcia krvn\u00fdch do\u0161ti\u010diek je 1,2 \u2013 1,5 x 1011, v perif\u00e9rnej krvi sa nach\u00e1dzaj\u00fa v priemernom po\u010dte 150 \u2013 400 x 109\/l a maj\u00fa objem 6 \u2013 12 fl(1). V panoptickom krvnom n\u00e1tere sa zobrazuj\u00fa do\u0161ti\u010dky ako ov\u00e1lne alebo okr\u00fahle objekty s priemernou ve\u013ekos\u0165ou 1,5 \u2013 3 \u00b5m. V ich strede sa nach\u00e1dzaj\u00fa \u010derveno-fialov\u00e9 granuly (granulom\u00e9ry) obklopen\u00e9 jemne bazofilnou cytoplazmou (hyalom\u00e9ra), ktor\u00e1 je ohrani\u010den\u00e1 plazmatickou membr\u00e1nou(2).<\/p>\n

Na povrchu membr\u00e1ny krvnej do\u0161ti\u010dky sa nach\u00e1dza ve\u013ek\u00e9 mno\u017estvo receptorov, ktor\u00e9 sl\u00fa\u017eia na zachytenie sign\u00e1lu, zabezpe\u010duj\u00fa jeho prenos a podie\u013eaj\u00fa sa na prestupe l\u00e1tok cez membr\u00e1nu. Popri be\u017en\u00fdch membr\u00e1nov\u00fdch receptoroch maj\u00fa na povrchu do\u0161ti\u010diek d\u00f4le\u017eit\u00fa \u00falohu glykoprote\u00edny. Zmeny \u0161trukt\u00fary a funkcie glykoprote\u00ednov krvn\u00fdch do\u0161ti\u010diek m\u00f4\u017eu sp\u00f4sobova\u0165 poruchy zr\u00e1\u017eania krvi(3). Biologick\u00e9 procesy regulovan\u00e9 do\u0161ti\u010dkami s\u00fa tie\u017e sprostredkovan\u00e9 membr\u00e1nov\u00fdmi glykoprote\u00ednmi (GP), ktor\u00e9 sa ozna\u010duj\u00fa r\u00edmskymi \u010d\u00edslicami, a ich podskupiny mal\u00fdmi arabsk\u00fdmi p\u00edsmenami. V s\u00fa\u010dasnosti je zn\u00e1mych viac ako 50 membr\u00e1nov\u00fdch glykoprote\u00ednov, pri\u010dom ka\u017ed\u00fd glykoprote\u00edn po obsaden\u00ed svojho v\u00e4zbov\u00e9ho miesta zabezpe\u010duje priebeh ur\u010dit\u00e9ho deja. Na membr\u00e1ne sa nach\u00e1dzaj\u00fa aj receptory sl\u00fa\u017eiace na naviazanie aktiva\u010dn\u00fdch a stimula\u010dn\u00fdch l\u00e1tok koagul\u00e1cie(4,5).<\/p>\n

P-selekt\u00edn, GMP-140 (CD62P) je membr\u00e1nov\u00fd glykoprote\u00edn gran\u00fal krvn\u00fdch do\u0161ti\u010diek, patr\u00ed k adhez\u00edvnym molekul\u00e1m. V pokojovom stave sa nach\u00e1dza v membr\u00e1nach \u03b1-gran\u00fal a v endoteli vo Weibelov\u00fdch-Paladeho telieskach. Po aktiv\u00e1cii je P-selekt\u00edn uvo\u013enen\u00fd z gran\u00fal. Pokojov\u00e9 do\u0161ti\u010dky exprimuj\u00fa asi 1 000 molek\u00fal P-selekt\u00ednu, aktivovan\u00fdch je viac ako 10 000 molek\u00fal. Exponovan\u00fd P-selekt\u00edn sa z\u00fa\u010dast\u0148uje na elimin\u00e1cii aktivovan\u00fdch krvn\u00fdch do\u0161ti\u010diek z cirkul\u00e1cie a sprostredkuje adh\u00e9ziu neutrofilov a monocytov na tromby a endotelov\u00e9 bunky(6,7).<\/p>\n

Prietokov\u00e1 cytometria je proces, ktor\u00fd umo\u017e\u0148uje kvalitat\u00edvnu a kvantitat\u00edvnu anal\u00fdzu optick\u00fdch a fluorescen\u010dn\u00fdch vlastnost\u00ed jednotliv\u00fdch buniek alebo in\u00fdch \u010dast\u00edc v suspenzii. Prietokov\u00fd cytometer sa sklad\u00e1 z fluidn\u00e9ho, optick\u00e9ho a po\u010d\u00edta\u010dov\u00e9ho syst\u00e9mu. V\u00fdsledkom anal\u00fdzy je v na\u0161om pr\u00edpade graficky zn\u00e1zornen\u00e1 (ko)expresia povrchov\u00fdch glykoprote\u00ednov v popul\u00e1cii krvn\u00fdch dosti\u010diek a dostupn\u00e1 s\u00fahrnn\u00e1 \u0161tatistika. S cie\u013eom pos\u00fadi\u0165 aktiv\u00e1ciu krvn\u00fdch do\u0161ti\u010diek m\u00f4\u017eeme pou\u017ei\u0165 testovanie povrchovej expresie aktiva\u010dn\u00e9ho znaku CD62P pomocou v\u00e4zby fluorescen\u010dne zna\u010denej monoklon\u00e1lnej protil\u00e1tky namierenej proti tomuto antig\u00e9nu, vizualizovanej prietokovou cytometriou.<\/p>\n

Le Minh a kolekt\u00edv sledovali prv\u00e9 pr\u00edznaky aktiv\u00e1cie trombocytov po indukcii ADP a porovnali dve met\u00f3dy zalo\u017een\u00e9 na morfologick\u00fdch a biochemick\u00fdch markeroch(8). Skenovacia elektr\u00f3nov\u00e1 mikroskopia krvn\u00fdch do\u0161ti\u010diek sa uskuto\u010d\u0148ovala paralelne s prietokovou cytometriou, aby sa kvantifikovala povrchov\u00e1 expresia P-selekt\u00ednu, akt\u00edvneho \u03b1IIb\u03b23-integr\u00ednu a fosfatidylser\u00ednu. Na z\u00e1klade z\u00edskan\u00fdch v\u00fdsledkov boli sledovan\u00e9 zmeny tvaru najcitlivej\u0161ie na n\u00edzku koncentr\u00e1ciu ADP v porovnan\u00ed s biochemick\u00fdmi markermi v nasleduj\u00facom po- rad\u00ed citlivosti: morfologick\u00e9 zmeny > v\u00e4zbov\u00e1 kapacita fibrinog\u00e9nu > expresia P-selekt\u00ednu > expoz\u00edcia fosfatidylser\u00ednu. Tieto v\u00fdsledky poukazuj\u00fa na vy\u0161\u0161iu citlivos\u0165 krvnej do\u0161ti\u010dky a v\u00fdhody vyu\u017eitia prietokovej cytometrie na detekciu povrchovej expresie P-selekt\u00ednu ako markera aktiv\u00e1cie krvn\u00fdch do\u0161ti\u010diek.<\/p>\n

Vyu\u017eitie prietokovej cytometrie zameran\u00e9 na \u0161t\u00fadium prokoagula\u010dn\u00fdch krvn\u00fdch do\u0161ti\u010diek v pokojov\u00fdch a agonistom stimulovan\u00fdch podmienkach v celej krvi a premyt\u00fdch krvn\u00fdch do\u0161ti\u010diek \u013eudsk\u00fdch a my\u0161\u00edch vzoriek opisuje vo svojej pr\u00e1ci Than a kol.(9).<\/p>\n

Vy\u0161etrenia glykoprote\u00ednov trombocytov maj\u00fa v\u00fdznam v posudzovan\u00ed stavov s trombocytovou hypo- a hyperreaktivitou a pri diferenci\u00e1lnej diagnostike trombocytop\u00e9ni\u00ed. Zn\u00ed\u017een\u00e1 expresia GPIIb\/IIIa, t. j. CD41\/CD61, sved\u010d\u00ed o Glanzmannovej trombast\u00e9nii, pri zn\u00ed\u017eenej expresii GPIb\/V\/IX, t. j. CD42b\/ CD42a, ide o Bernardov-Soulierov syndr\u00f3m. V uveden\u00fdch pr\u00edpadoch ide o heredit\u00e1rne trombocytop\u00e9nie, ale vy\u0161etrenie glykoprote\u00ednov trombocytov sa odpor\u00fa\u010da aj pri stavoch s ich hyperreaktivitou (aterosklerotick\u00e9 cievne ochorenia, diabetes mellitus, arteri\u00e1lne a ven\u00f3zne tromb\u00f3zy, umel\u00e9 chlopne, chronick\u00e9 z\u00e1palov\u00e9 \u010drevn\u00e9 ochorenia, syst\u00e9mov\u00e9 ochorenia) alebo hyporeaktivitou (vroden\u00e9 defekty receptorov trombocytov, ren\u00e1lne a hepat\u00e1lne insuficiencie, esenci\u00e1lne trombocyt\u00e9mie. V\u00e4\u010d\u0161inou sa hodnotia zmeny adhez\u00edvnych receptorov ako GPIb, t. j. CD42b\/CD42a, GPIIb\/GPIIIa, t. j. CD41\/ CD61, a aktiva\u010dn\u00fdch markerov GMP 140, t. j. CD62P, GP53, j. CD63, trombospond\u00ednov\u00fd receptor CD36(10).<\/p>\n

 <\/p>\n

Materi\u00e1l a metodika<\/strong><\/p>\n

 <\/p>\n

Pr\u00edprava vzorky<\/strong><\/p>\n

Vzorku krvi na anal\u00fdzu do\u0161ti\u010dkov\u00fdch glykoprote\u00ednov sme pacientom odobrali do jednorazovej vakutajnerovej sk\u00famavky s antikoagulantom 3,2 % (0,109 mol\/l) citr\u00f3nanom sodn\u00fdm. Sk\u00famavku s krvou sme centrif\u00fagovali pri 200 g 10 min\u00fat.<\/p>\n

PRP sme oddelili od krviniek do inej sk\u00famavky.<\/p>\n

 <\/p>\n

Met\u00f3da<\/strong><\/p>\n

Do\u0161ti\u010dkov\u00e9 glykoprote\u00edny sme analyzovali na prietokovom cytometri FACSVerse (Becton Dickinson and company, San Jose, USA). Pre povrchov\u00fa imunofenotypiz\u00e1ciu sme pou\u017eili techniku dvojit\u00e9ho priameho zna\u010denia fluorinovan\u00fdmi monoklon\u00e1lnymi protil\u00e1tkami: anti-CD42a\/FITC (clone SZ1, Beckman Coulter, San Diego, USA), anti-CD62P\/PE (clone AC1.2, Becton Dickinson).<\/p>\n

Na\u0161\u00edm cie\u013eom bolo zisti\u0165, \u010di met\u00f3da stanovenia do\u0161ti\u010dkov\u00fdch glykoprote\u00ednov v PRP, ktor\u00e1 je pova\u017eovan\u00e1 za zlat\u00fd \u0161tandard, ovplyv\u0148uje expresiu CD62P na do\u0161ti\u010dk\u00e1ch. Vych\u00e1dzali sme z predpokladu, \u017ee plazmu bohat\u00fa na do\u0161ti\u010dky pripravujeme centrifug\u00e1ciou celej krvi, \u010d\u00edm m\u00f4\u017eeme sp\u00f4sobi\u0165 \u010diasto\u010dn\u00fa aktiv\u00e1ciu do\u0161ti\u010diek. Ka\u017ed\u00e9mu z 50 zdrav\u00fdch darcov krvi bola stanoven\u00e1 expresia CD62P v celej krvi a s\u00fa\u010dasne v PRP. Ako pozit\u00edvnu kontrolu sme pou\u017eili aktivovan\u00e9 krvn\u00e9 do\u0161ti\u010dky, ktor\u00e9 sme aktivovali ADP s fin\u00e1lnou koncentr\u00e1ciou 10 \u00b5M (CD42b-FITC\/CD62P-PE). Ako negat\u00edvnu kontrolu sme pou\u017eili izotypov\u00fa kontrolu IgG1\/IgG2 (FITC\/PE).<\/p>\n

 <\/p>\n

V\u00fdsledky<\/strong><\/p>\n

Obr\u00e1zok 1 <\/strong>ukazuje sp\u00f4sob anal\u00fdzy a rozdiel medzi expresiou CD62P na do\u0161ti\u010dk\u00e1ch charakterizovan\u00fdch pomocou rozptylov\u00fdch parametrov (FSC a SSC, oblas\u0165 \u201edo\u0161ti\u010dky\u201c) a expresiou antig\u00e9nu CD42a v celej krvi (A, C) a v PRP (B, D). \u010c\u00edsla v jednotliv\u00fdch kvadrantoch \u0161tatisticky v C a D vyjadruj\u00fa percentu\u00e1lne zast\u00fapenie jednotliv\u00fdch popul\u00e1ci\u00ed objektov z oblasti \u201edo\u0161ti\u010dky\u201c, zast\u00fapenie CD62P \u2013 pozit\u00edvnych do\u0161ti\u010diek bolo ur\u010den\u00e9 ako 100 % x (%Q2)\/(%Q2 + %Q4). V pr\u00edpade uvedenom na obr\u00e1zku \u010d. 1 sp\u00f4sobila pr\u00edprava PRP zdvojn\u00e1sobenie zast\u00fapenia CD42+CD62P+ do\u0161ti\u010diek v PRP vzorke (16,7 %) v porovnan\u00ed so vzorkou celej krvi (8,5 %).<\/p>\n

 <\/p>\n

Diskusia<\/strong><\/p>\n

Jedn\u00fdm z cie\u013eov pr\u00e1ce bolo porovna\u0165 dve met\u00f3dy anal\u00fdzy krvn\u00fdch do\u0161ti\u010diek, a to stanovenie expresie do\u0161ti\u010dkov\u00fdch glykoprote\u00ednov v celej krvi a v PRP u zdrav\u00fdch darcov (v kontrolnej skupine).<\/p>\n

Porovnan\u00edm oboch met\u00f3d na z\u00e1klade stanovenia koexpresie CD42a\/CD62P zn\u00e1zornen\u00e9 na obr\u00e1zku 1 <\/strong>sme zistili \u0161tatistick\u00fa v\u00fdznamnos\u0165 CD42a\/CD62P v PRP oproti celej krvi (P < 0,0005; graf 1<\/strong>).<\/p>\n

Na z\u00e1klade z\u00edskan\u00fdch \u0161tatistick\u00fdch v\u00fdsledkov sa jav\u00ed met\u00f3da stanovenia do\u0161ti\u010dkov\u00fdch glykoprote\u00ednov v celej krvi praktickej\u0161ia a \u0161etrnej\u0161ia pre krvn\u00e9 do\u0161ti\u010dky ne\u017e met\u00f3da stanovenia v PRP. Porovnan\u00edm anal\u00fdz expresie CD42a-FITC\/ CD62P-PE v celej krvi a v PRP v kontrolnej skupine sme zistili, \u017ee izol\u00e1cia PRP vpl\u00fdva na aktiv\u00e1ciu do\u0161ti\u010diek do takej miery, \u017ee rozdiel je oproti met\u00f3de v celej krvi \u0161tatisticky v\u00fdznamn\u00fd s hodnotou P \u02c2 0,0005. Expresia CD62P na do\u0161ti\u010dk\u00e1ch v PRP je v porovnan\u00ed s expresiou CD62P na do\u0161ti\u010dk\u00e1ch v celej krvi \u0161tatisticky v\u00fdznamne zv\u00fd\u0161en\u00e1.<\/p>\n

 <\/p>\n

Z\u00e1ver<\/strong><\/p>\n

Porovnan\u00edm anal\u00fdz expresie CD42a-FITC\/CD62P-PE v celej krvi a v PRP v kontrolnej skupine sme zistili, \u017ee izol\u00e1cia PRP vpl\u00fdva na aktiv\u00e1ciu do\u0161ti\u010diek do takej miery, \u017ee rozdiel je oproti met\u00f3de v celej krvi \u0161tatisticky v\u00fdznamn\u00fd s hodnotou P \u02c2 0,0005. Expresia CD62P na do\u0161ti\u010dk\u00e1ch v PRP je v porovnan\u00ed s expresiou CD62P na do\u0161ti\u010dk\u00e1ch v celej krvi \u0161tatisticky v\u00fdznamne zv\u00fd\u0161en\u00e1.<\/p>\n

Pou\u017eit\u00edm met\u00f3dy anal\u00fdzy do\u0161ti\u010dkov\u00fdch glykoprote\u00ednov v celej krvi sa pribl\u00ed\u017eime podmienkam in vivo a minimalizujeme aktiv\u00e1ciu do\u0161ti\u010diek.<\/p>\n

 <\/p>\n

Po\u010fakovanie<\/em><\/strong><\/p>\n

T\u00e1to pr\u00e1ca bola podporen\u00e1 projektami APVV \u2013 16-0020, Vega 1\/0168\/16, Vega 1\/0187\/17 a Centra excelentnosti pre perinatologick\u00fd v\u00fdskum (CEPV II, ITMS 26220120036) a Centra excelentnosti pre v\u00fdskum v personalizovanej terapii (CEVYPET).<\/em><\/p>\n

 <\/p>\n

LITERAT\u00daRA<\/strong><\/p>\n

    \n
  1. Penka M a kol. Hematologie a transfuzn\u00ed l\u00e9ka\u0159stv\u00ed I. Grada Publishing, s., 2011, 33.<\/li>\n
  2. Sakalov\u00e1 A a kol. Hematol\u00f3gia a transf\u00faziol\u00f3gia, Vydavate\u013estvo Osveta, 1995, 102-103.<\/li>\n
  3. Nos\u00e1\u013e R, Jan\u010dinov\u00e1 V. Krvn\u00e9 do\u0161ti\u010dky v biol\u00f3gii a medic\u00edne. Veda, Vydavate\u013estvo Slovenskej akad\u00e9mie vied, Bratislava, 1990, 15-16.<\/li>\n
  4. Evans WH, Graham Memrane structure and function. Oxford, England: Oxford IRL Press, Oxford University Press, 1988, 11-29.<\/li>\n
  5. Sta\u0161ko J, Barto\u0161ov\u00e1 L, M\u00fdtnik M, Kubisz P. Are the platelets activated in sticky platelet syndrome? Thromb Res, 2011, 128: 96-97.<\/li>\n
  6. Curvers J, de Wildt-Eggen J, Heeremans J, et Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort. Transfusion. 2008 Jul;48(7):1439-46.<\/li>\n
  7. Vu\u010deti\u0107 D, Ili\u0107 V, Vojvodi\u0107 D, et al. Flow cytometry analysis of platelet populations: usefulness for monitoringthe storage lesion in pooled buffycoat platelet Blood Transfus. 2018 Jan;16(1):83-92.<\/li>\n
  8. Le Minh G, Peshkova AD, Andrianova IA, et Differential sensitivity of various markers of platelet activation with adenosine diphosphate.Bionanoscience. 2019 marec; 9 (1): 53-58. Epub 2018, 10. decembra.<\/li>\n
  9. Tan CW, Bourcy M, Pasalic L, Chen VM. Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe. Methods Mol Bi- 2019;1967:305-321.<\/li>\n
  10. Kubisz P a Hematol\u00f3gia a transf\u00faziol\u00f3gia, Grada Publishing, a. s., 2006, 257-260.<\/li>\n<\/ol>\n

     <\/p>\n","protected":false},"excerpt":{"rendered":"

    *A rare case of autochthonous human dirofilariasis with the manifestation of pseudotumor of the epididymis caused by helminth Dirofilaria repens   Krvn\u00e9 do\u0161ti\u010dky s\u00fa bezjadrov\u00e9 \u00falomky cytoplazmy vznikaj\u00face z megakaryocytov v kostnej dreni. Denn\u00e1 produkcia krvn\u00fdch do\u0161ti\u010diek je 1,2 \u2013 1,5 x 1011, v perif\u00e9rnej krvi sa nach\u00e1dzaj\u00fa v priemernom po\u010dte 150 \u2013 400 x<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[294],"tags":[1723,1722],"class_list":["post-2239","post","type-post","status-publish","format-standard","hentry","category-hematology","tag-flow-cytometry","tag-platelet-glycoproteins","typ_clanku-original-work"],"acf":{"abstrakt":"

    The membrane of a platelet is rich in receptors mediating signal sensing and transmission and participating in substance transport across the membrane. Flow cytometry can measure the circulation of non-activated as well as activated platelets by qualitative and\/or quantitative analysis of surface markers expressed at a certain level during the life cycle of platelets. We aimed to determine whether the method of measurement of the platelet glycoproteins expression in the platelet-rich plasma (PRP) results in altered expression of the CD62P activation antigen on the platelet surface. In this article, we present our own experience. We compare platelet glycoprotein analysis in whole blood and PRP.<\/p>\n

    Keywords: <\/strong>platelet glycoproteins, flow cytometry<\/p>\n","casopis":[{"ID":2233,"post_author":"7","post_date":"2021-09-19 22:32:27","post_date_gmt":"2021-09-19 20:32:27","post_content":"We\u2019re bringing you this year\u2019s 1st<\/sup> issue of newslab, the scientific journal<\/strong>\r\n\r\n \r\n

      \r\n \t
    • Genomic variability of virus Sars-CoV-2<\/li>\r\n \t
    • Contribution of immunohistochemistry to histomorphological diagnostics of COVID-19 pneumonia<\/li>\r\n \t
    • Detection of microsatellite instability in Lynch syndrome-associated tumours<\/li>\r\n \t
    • Comparison of platelet glycoprotein analysis in the wholeh blood and plasma rich in platelets<\/li>\r\n \t
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