{"id":1196,"date":"2016-12-06T22:40:45","date_gmt":"2016-12-06T21:40:45","guid":{"rendered":"http:\/\/www.newslab.sk\/2016\/12\/06\/vyuzitie-prietokovej-cytometrie-pri-stanoveni-prognostickych-markerov-cd38-a-zap-70-u-pacientov-s-b-cll\/"},"modified":"2017-10-04T14:53:28","modified_gmt":"2017-10-04T12:53:28","slug":"application-of-flow-based-cytometry-in-establishing-prognostic-cd38-and-zap-70-markers-in-patients-with-b-cll","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/application-of-flow-based-cytometry-in-establishing-prognostic-cd38-and-zap-70-markers-in-patients-with-b-cll\/","title":{"rendered":"Application of Flow-based Cytometry in Establishing Prognostic CD38 and ZAP-70 Markers in Patients with B-CLL"},"content":{"rendered":"

*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf\u00a0<\/strong><\/span>
\nattachment at the end of the paper.<\/strong><\/span><\/p>\n

 <\/p>\n

Chronick\u00e1 lymfocytov\u00e1 leuk\u00e9mia<\/strong> z\u00a0B lymfocytov (B-CLL) je definovan\u00e1 ako n\u00edzkoagres\u00edvne lymfoproliferat\u00edvne ochorenie zapr\u00ed\u010dinen\u00e9 klon\u00e1lnou prolifer\u00e1ciou mal\u00edgne transformovan\u00fdch mal\u00fdch zrel\u00fdch B lymfocytov. Tieto bunky so \u0161pecifick\u00fdm imunofenotypom sa akumuluj\u00fa v\u00a0perif\u00e9rnej krvi, kostnej dreni, lymfatick\u00fdch uzlin\u00e1ch, pr\u00edpadne v in\u00fdch hemopoetick\u00fdch a\u00a0non-hemopoetick\u00fdch org\u00e1noch. Pr\u00ed\u010dinou n\u00e1rastu po\u010dtu lymfocytov nie je len ich nadmern\u00e1 klon\u00e1lna prolifer\u00e1cia, ale aj porucha apopt\u00f3zy (1). Pod\u013ea krit\u00e9ri\u00ed medzin\u00e1rodn\u00e9ho konsenzu o\u00a0B-CLL (tzv. krit\u00e9ri\u00e1 IWCLL) je ochorenie definovan\u00e9 pr\u00edtomnos\u0165ou monoklonovej lymfocyt\u00f3zy v\u00a0perif\u00e9rnej krvi nad 5 x 109<\/sup>\/l, trvaj\u00facou najmenej 3 mesiace a spolo\u010dne so\u00a0stanoven\u00edm typick\u00e9ho imunofenotypov\u00e9ho profilu patologick\u00fdch buniek prietokovou cytometriou s\u00fa dostato\u010dn\u00fdmi krit\u00e9riami na diagnostiku B-CLL (2). WHO klasifik\u00e1cia sa opiera o laborat\u00f3rnu diagnostiku B lymfocytov (morfologick\u00e9, imunofenotypov\u00e9, genetick\u00e9, ako aj\u00a0molekulovo-biologick\u00e9 parametre) a\u00a0klinick\u00e9 prejavy ochorenia.<\/p>\n

B-CLL\u00a0tvor\u00ed cca 25 % \u2013 30 % z leuk\u00e9mi\u00ed dospel\u00fdch v\u00a0Eur\u00f3pe a\u00a0Severnej Amerike, \u010d\u00edm sa zara\u010fuje k\u00a0naj\u010dastej\u0161ie sa vyskytuj\u00facim leuk\u00e9mi\u00e1m (1). Ke\u010f\u017ee je roky bezpr\u00edznakov\u00e1, \u010dasto sa diagnostikuje iba n\u00e1hodne (asi 50 % pr\u00edpadov) pri vy\u0161etren\u00ed parametrov krvn\u00e9ho obrazu.<\/p>\n

B-CLL sa vyzna\u010duje ve\u013ekou heterogenitou a\u00a0variabiln\u00fdm priebehom ochorenia, ktor\u00fd rozde\u013euje pacientov na skupinu s\u00a0indolentn\u00fdm charakterom a\u00a0na skupinu s\u00a0agres\u00edvnym, r\u00fdchlo progreduj\u00facim ochoren\u00edm a\u00a0kr\u00e1tkodob\u00fdm pre\u017e\u00edvan\u00edm (3). Snaha opiera\u0165 sa pri mana\u017emente terapie o\u00a0vybran\u00e9 parametre s\u00a0prognostick\u00fdm v\u00fdznamom vypl\u00fdva z\u00a0biologickej nehomog\u00e9nnosti B-CLL rozpoznate\u013enej na morfologickej, fenotypovej a\u00a0genetickej \u00farovni. Koncom 90. rokov 20. storo\u010dia vst\u00fapili pri \u0161t\u00fadiu biologickej heterogenity B-CLL do popredia genetick\u00e9 a\u00a0molekulovo-biologick\u00e9 parametre, ktor\u00e9\u00a0umo\u017enili rozdelenie dovtedy zn\u00e1mych prognostick\u00fdch markerov na klasick\u00e9<\/strong> (star\u00e9, respekt\u00edve klinick\u00e9) a\u00a0na molekulov\u00e9<\/strong> (nov\u00e9, modern\u00e9) (4).<\/p>\n

K\u00a0nov\u00fdm prognostick\u00fdm markerom zara\u010fujeme muta\u010dn\u00fd stav IgVH <\/sub>g\u00e9nov, expresiu CD38, expresiu ZAP-70,\u00a0chromoz\u00f3mov\u00e9 abnormality (del17p, del11q, del 6q, triz\u00f3mia 12 a\u00a0del 13q)<\/p>\n

a\u00a0mut\u00e1ciu g\u00e9nu TP53. Pr\u00edtomnos\u0165 somatick\u00fdch mut\u00e1ci\u00ed v\u00a0hypervariabiln\u00fdch \u00fasekoch (IgVH<\/sub>) g\u00e9nov pre \u0165a\u017ek\u00e9 re\u0165azce imunoglobul\u00ednov umo\u017enila zadefinova\u0165 progn\u00f3zu a\u00a0d\u013a\u017eku pre\u017e\u00edvania pacientov. Nemutovan\u00fd stav IgVH <\/sub>g\u00e9nov (homol\u00f3gia vo > 98,0 % sekvenci\u00ed) koreluje s\u00a0nepriaznivou progn\u00f3zou a krat\u0161\u00edm medi\u00e1nom pre\u017e\u00edvania a\u00a0naopak, za prognosticky priazniv\u00fd stav sa pova\u017euje pr\u00edtomnos\u0165 somatick\u00fdch mut\u00e1ci\u00ed vo \u2265 2,0 % sekvenci\u00ed (4).<\/p>\n

Stanovenie muta\u010dn\u00e9ho stavu IgVH<\/sub> je v\u0161eobecne pova\u017eovan\u00e9 za technologicky, \u010dasovo i finan\u010dne n\u00e1ro\u010dn\u00e9 a v s\u00fa\u010dasnosti \u0165a\u017eko realizovate\u013en\u00e9 v be\u017en\u00fdch laborat\u00f3rnych podmienkach pre n\u00e1ro\u010dnos\u0165 spracovania materi\u00e1lu (5).<\/p>\n

Expresia CD38<\/strong> bola prv\u00fdm markerom, pri ktorom sa preuk\u00e1zala korel\u00e1cia s IgVH<\/sub> statusom. CD38 je 45kDa transmembr\u00e1nov\u00fd glykoprote\u00edn, ktor\u00fd p\u00f4sob\u00ed s\u00fa\u010dasne ako enz\u00fdm a receptor. Vy\u0161etrenie expresie CD38 na povrchu leukemick\u00fdch buniek prietokovou cytometriou je v klinick\u00fdch laborat\u00f3ri\u00e1ch relat\u00edvne dostupn\u00e9, av\u0161ak jeho pou\u017eitie ako prognostick\u00e9ho markera pri\u00a0B-CLL sa postupom \u010dasu uk\u00e1zalo ako nejednozna\u010dn\u00e9. Predov\u0161etk\u00fdm sa nepotvrdila absol\u00fatna korel\u00e1cia expresie s IgVH<\/sub> muta\u010dn\u00fdm statusom a objavili sa aj r\u00f4zne n\u00e1zory na stabilitu expresie antig\u00e9nu CD38 a na ur\u010denie cut-off hodnoty pre pozitivitu CD38 na leukemick\u00fdch bunk\u00e1ch z\u00a0prognostick\u00e9ho h\u013eadiska (hodnoty od 7 % a\u017e do 20 %, respekt\u00edve 30 %) (6). V\u00a0s\u00fa\u010dasnosti sa za klinicky relevantn\u00fa mieru expresie pova\u017euje arbitr\u00e1rne (\u0161tatisticky) ur\u010den\u00e1 hodnota v klinick\u00fdch laborat\u00f3ri\u00e1ch \u2265 30 % detegovan\u00e1 prietokovou cytometriou (4). \u010cas\u0165 pacientov vykazuje bimod\u00e1lny profil expresie, najbe\u017enej\u0161ie sa pozoruje zmena CD38 negativity na CD38 pozitivitu, ktor\u00e1 sa \u010dasto sp\u00e1ja s\u00a0progresiou ochorenia (7).<\/p>\n

Expresia prote\u00ednu ZAP-70<\/strong> patr\u00ed medzi \u010fal\u0161ie prognosticky v\u00fdznamn\u00e9 parametre, o ktor\u00fdch sa uva\u017eovalo, \u017ee nahradia stanovenie IgVH<\/sub>. ZAP-70, zeta asociovan\u00fd prote\u00edn s\u00a0molekulovou hmotnos\u0165ou 70 kDa, je k\u00f3dovan\u00fd g\u00e9nom na dlhom ramienku 2. chromoz\u00f3mu v 2q2 lokuse (8). T\u00e1to tyroz\u00edn kin\u00e1za z\u00a0rodiny tzv. Syk prote\u00ednov obsahuje dve termin\u00e1lne SH2 dom\u00e9ny a C-termin\u00e1lnu kin\u00e1zov\u00fa dom\u00e9nu. Miestom prim\u00e1rnej expresie prote\u00ednu ZAP-70 je cytoplazma T a NK lymfocytov (9). ZAP-70 prote\u00edn bol identifikovan\u00fd ako fosforyla\u010dn\u00fd partner zeta re\u0165azca CD3 komplexu asociovan\u00e9ho s\u00a0TCR (T bunkov\u00fd receptor) komplexom na T lymfocytoch. Aktiv\u00e1cia T lymfocytov prostredn\u00edctvom sign\u00e1lu odovzd\u00e1van\u00e9ho cez TCR receptor je nevyhnutn\u00e1 na v\u00fdvoj imunitnej odpovede, z\u00a0\u010doho vypl\u00fdva, \u017ee ZAP-70 zohr\u00e1va k\u013e\u00fa\u010dov\u00fa \u00falohu v TCR signaliz\u00e1cii. Deficit ZAP-70 m\u00e1 za n\u00e1sledok absenciu maturovan\u00fdch CD8+ lymfocytov, blokuje aktiv\u00e1ciu CD4+ T lymfocytov sprostredkovan\u00fa cez TCR a\u00a0m\u00f4\u017ee sp\u00f4sobi\u0165 autozom\u00e1lne dedi\u010dn\u00fa formu \u0165a\u017ek\u00e9ho kombinovan\u00e9ho deficitu \u0161pecifickej imunity (Severe Combined Immunodeficiency \u2013 SCID) (10). Prote\u00edn ZAP-70 exprimuj\u00fa v r\u00f4znej miere aj B lymfocyty a n\u00e1dorov\u00e9 bunky viacer\u00fdch typov B NHL vr\u00e1tane B-CLL (8). Expresia prote\u00ednu ZAP-70 koreluje s\u00a0nemutovan\u00fdm stavom IgVH<\/sub> g\u00e9nov, a teda pozit\u00edvna expresia ZAP-70 predstavuje nepriazniv\u00fa progn\u00f3zu (9). Pri B-CLL ZAP-70 pravdepodobne zosil\u0148uje sign\u00e1lne dr\u00e1hy sprostredkovan\u00e9 receptorom B lymfocytov, \u010do je proces prispievaj\u00faci k\u00a0hor\u0161ej progn\u00f3ze u ZAP-70 pozit\u00edvnych pacientov. Podobne ako pri T lymfocytoch, m\u00e1 ZAP-70 v\u00fdznam pre chemok\u00ednov\u00fa stimul\u00e1ciu buniek B-CLL. Predpoklad\u00e1 sa, \u017ee expresia ZAP-70 by mohla vies\u0165 k migr\u00e1cii leukemick\u00fdch buniek do mikroprostredia v\u00fdhodn\u00e9ho na pre\u017eitie \u010di prolifer\u00e1ciu. Av\u0161ak korel\u00e1cia medzi expresiou ZAP-70 a\u00a0muta\u010dn\u00fdm stavom IgVH <\/sub>g\u00e9nov nie je absol\u00fatna, a\u00a0teda pr\u00edtomnos\u0165 diskordantn\u00fdch n\u00e1lezov (v\u00a0z\u00e1vislosti od pou\u017eitej met\u00f3dy detekcie od 8 % do 25 %) potvrdzuje ZAP-70 ako nez\u00e1visl\u00fd prognostick\u00fd faktor (4). Pri porovnan\u00ed vz\u00e1jomnej korel\u00e1cie IgVH<\/sub> a CD38, respekt\u00edve ZAP-70, je \u0161tatisticky v\u00fdznamnej\u0161ia korel\u00e1cia IgVH<\/sub> a ZAP-70 (11, 12). Ke\u010f\u017ee ZAP-70 a\u00a0CD38 vz\u00e1jomne nekoreluj\u00fa, ich s\u00fa\u010dasn\u00e9 stanovenie m\u00e1 v\u00e4\u010d\u0161iu v\u00fdpovedn\u00fa hodnotu pri identifik\u00e1cii pacientov s\u00a0r\u00f4znou progn\u00f3zou ne\u017e anal\u00fdza len jedn\u00e9ho izolovan\u00e9ho parametra. Kombinovan\u00e1 anal\u00fdza CD38 a\u00a0ZAP-70 umo\u017e\u0148uje identifikova\u0165 jednak skupinu pacientov s\u00a0dobrou progn\u00f3zou, ktor\u00ed vykazuj\u00fa fenotyp CD38-\/ZAP-70-, skupinu s\u00a0nepriaznivou progn\u00f3zou (fenotyp CD38+\/ZAP-70+), ako aj pacientov s\u00a0diskordantn\u00fdm fenotypom CD38-\/ZAP-70+, respekt\u00edve CD38+\/ZAP-70- a\u00a0stredn\u00fdm rizikom (13).<\/p>\n

Na stanovenie ZAP-70<\/strong> sa v\u00a0s\u00fa\u010dasnosti vyu\u017e\u00edvaj\u00fa imunohistochemick\u00e9 (semikvantitat\u00edvne hodnotenie) a imunocytochemick\u00e9 met\u00f3dy, Western-blotting, anal\u00fdza mRNA a tie\u017e prietokov\u00e1 cytometria (8). Interpret\u00e1ciu anal\u00fdzy pomocou Western-blotingu komplikuje pr\u00edpadn\u00e1 kontamin\u00e1cia vzorky T lymfocytmi, \u010do m\u00f4\u017ee vies\u0165 k\u00a0falo\u0161ne pozit\u00edvnym v\u00fdsledkom (14). Preto sa do popredia dost\u00e1va snaha o\u00a0zavedenie spo\u013eahlivej, rutinnej a relat\u00edvne jednoduchej met\u00f3dy pomocou prietokovej cytometrie.<\/p>\n

Bolo vypracovan\u00fdch nieko\u013eko protokolov na detekciu ZAP-70, ako aj\u00a0odpor\u00fa\u010dan\u00ed na interpret\u00e1ciu v\u00fdsledku expresie ZAP-70 (tabu\u013eka 1). Detekciu expresie ZAP-70 prietokovou cytometriou komplikuj\u00fa viacer\u00e9 probl\u00e9my, ku ktor\u00fdm sa zara\u010fuje predov\u0161etk\u00fdm slab\u00e1 expresia ZAP-70 v\u00a0cytoplazme patologick\u00fdch B lymfocytov a ch\u00fdbanie jednotne pou\u017e\u00edvanej \u0161tandardizovanej met\u00f3dy (3, 16).<\/p>\n

Popri met\u00f3dach so separovan\u00fdmi mononukle\u00e1rnymi bunkami s\u00fa vypracovan\u00e9 protokoly s imunofenotypovou anal\u00fdzou plnej krvi. Pracovn\u00e9 postupy sa \u010falej odli\u0161uj\u00fa v\u00a0pou\u017e\u00edvan\u00ed r\u00f4znych klonov anti-ZAP-70 protil\u00e1tok priamou, respekt\u00edve nepriamou imunofluorescen\u010dnou met\u00f3dou. Pomerne ve\u013ek\u00e1 variabilita je aj vo fixa\u010dno-permeabiliza\u010dn\u00fdch met\u00f3dach, pou\u017e\u00edvan\u00ed r\u00f4znopo\u010detn\u00fdch multifarebn\u00fdch protokolov a\u00a0analytickej strat\u00e9gii (tvorba logick\u00fdch \u201egate\u201c, ur\u010denie cut-off pozitivity) (tabu\u013eka 1).<\/p>\n

Pri interpret\u00e1cii v\u00fdsledkov anal\u00fdzy sa autori opieraj\u00fa o\u00a0vyjadrenie pozitivity expresie ZAP-70 v\u00a0percent\u00e1ch, alebo vyjadruj\u00fa pomern\u00fa priemern\u00fa intenzitu fluorescencie (MFI). Expresiu ZAP-70 sa deteguje v\u00a0percent\u00e1ch vo vz\u0165ahu k\u00a0externej negat\u00edvnej, izotypovej kontrole, internej pozit\u00edvnej kontrole (T lymfocyty, NK lymfocyty), internej negat\u00edvnej kontrole (CD5- B lymfocyty) alebo negat\u00edvnej kontrole zdrav\u00fdch kontrol, kde sa cut-off pozitivity ZAP-70 pohybuje v\u00a0rozmedz\u00ed 10 % a\u017e 30 %. Pri pomernom vyjadren\u00ed MFI sa pozitivita expresie ZAP-70 stanov\u00ed koeficientom MFI CD3+ T lymfocytov k MFI CD5+ B lymfocytov (T\/CLL B), podielom MFI CD5+ B lymfocytov vo\u010di CD5- B lymfocytom (CLL B\/B Ly), respekt\u00edve T lymfocytom (CLL B\/T) (6, 9, 12, 15, 17 \u2013 23).<\/p>\n

V\u00a0porovnan\u00ed s IgVH <\/sub>je cytometrick\u00e9 stanovenie CD38 a\u00a0ZAP-70 menej finan\u010dne a\u00a0\u010dasovo n\u00e1ro\u010dn\u00e9 (9). Prietokov\u00e1 cytometria sa stala met\u00f3dou vo\u013eby aj pre svoju relat\u00edvnu jednoduchos\u0165 a\u00a0dostupnos\u0165. Umo\u017e\u0148uje \u0161pecifick\u00e9 ohrani\u010denie cie\u013eovej popul\u00e1cie buniek a\u00a0odl\u00ed\u0161enie expresie ZAP-70 na patologickom klone B lymfocytov\u00a0od v\u00fdraznej pozitivity ZAP-70 v\u00a0cytoplazme T a\u00a0NK lymfocytov. Expresia ZAP-70 m\u00f4\u017ee by\u0165 relevantnej\u0161\u00edm prognostick\u00fdm indik\u00e1torom definuj\u00facim B-CLL s\u00a0hor\u0161ou progn\u00f3zou pri nemutovanom IgVH <\/sub>statuse.<\/p>\n

Po\u010fakovanie:<\/em><\/strong> Pr\u00e1ca bola podporen\u00e1 projektom ITMS 26220220197 spolufinancovan\u00fdm zo zdrojov E\u00da (Modern\u00e9 technol\u00f3gie monitorovania a\u00a0diagnostiky ochoren\u00ed ohrozuj\u00facich verejn\u00e9 zdravie). <\/em><\/p>\n

\u201eTento \u010dl\u00e1nok vznikol na z\u00e1klade v\u00fdskumn\u00fdch aktiv\u00edt v projekte, ktor\u00fd bol podporen\u00fd v r\u00e1mci OP V\u00fdskum a v\u00fdvoj, s n\u00e1zvom: V\u00fdskumn\u00e9 centrum modern\u00fdch technol\u00f3gi\u00ed monitorovania a diagnostiky ochoren\u00ed ohrozuj\u00facich verejn\u00e9 zdravie, ITMS 26220220197, spolufinancovan\u00fd zo zdrojov Eur\u00f3pskeho fondu region\u00e1lneho rozvoja.\u201c<\/em><\/strong><\/p>\n

 <\/p>\n

Tabu\u013eka 1.<\/em><\/strong> Porovnanie niektor\u00fdch metod\u00edk na stanovenie ZAP-70 prietokovou cytometriou<\/p>\n\n\n\n\n\n\n\n\n\n\n
Met\u00f3da<\/strong><\/td>\nanti-ZAP-70<\/strong><\/td>\nFix\u00e1cia\/<\/strong><\/p>\n

permeabiliz\u00e1cia<\/strong><\/td>\n

Anal\u00fdza<\/strong><\/td>\nCut-off<\/strong><\/td>\nAutor<\/strong><\/td>\n<\/tr>\n
Priama IF<\/td>\n1E7.2 Alexa-488<\/p>\n

Caltag<\/td>\n

Fix and Perm, Caltag<\/td>\nIzotypov\u00e1. vs.<\/p>\n

T\/NK kontrola<\/td>\n

20 %<\/td>\nZucchetto et al., 2006 (20)<\/td>\n<\/tr>\n
Nepriama<\/p>\n

IF<\/td>\n

2F3.2 Upstate<\/td>\nParaformaldehyd-Tween<\/td>\nIzotypov\u00e1 kontrola<\/td>\n10 %<\/td>\nOrchard et al., 2004 (12)<\/td>\n<\/tr>\n
Priama<\/p>\n

IF<\/td>\n

1E7.2 Alexa-488<\/p>\n

BD<\/td>\n

Paraformaldehyd-Saponin<\/td>\nIzotypov\u00e1 kontrola<\/td>\n20 %<\/td>\nRasenti et al., 2004 (11)<\/td>\n<\/tr>\n
Priama<\/p>\n

IF<\/td>\n

1E7.2PE<\/p>\n

Caltag<\/td>\n

Fix and Perm, Caltag<\/td>\nT\/NK kontrola<\/td>\n20 %<\/td>\nD\u00b4Arena et al., 2007 (13)<\/td>\n<\/tr>\n
Priama<\/p>\n

IF<\/td>\n

136F12 Alexa 647 Cell Signaling<\/td>\nIntraprep, Beckman-Coulter<\/td>\nIzotypov\u00e1 kontrola<\/td>\n25 %<\/td>\nSlack et al., 2007 (24)<\/td>\n<\/tr>\n
Priama<\/p>\n

IF<\/td>\n

SBZAP PE, BC<\/td>\nFormaldehyd\/Triton X-100<\/td>\nMFI<\/td>\n<\/td>\nShankey et al., 2006 (17)<\/td>\n<\/tr>\n
Nepriama<\/p>\n

IF<\/td>\n

2F3.2 Upstate<\/td>\nFix and Perm, Caltag<\/td>\nT\/NK kontrola<\/td>\n20 %<\/td>\nCrespo et al., 2003 (9)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

IF \u2013 imunofluorescen\u010dn\u00e1 met\u00f3da, BD \u2013 BectonDickinson, BC \u2013 BeckmanCoulter, MIF \u2013 Mean Fluorescence Intensity<\/p>\n

 <\/p>\n

Literat\u00fara<\/strong><\/p>\n

    \n
  1. Adam Z, Vorl\u00ed\u010dek J, et al. Hematologie II: P\u0159ehled mal\u00edgn\u00edch hematologick\u00fdch nemoc\u00ed. <\/em>vyd. Praha, \u010cesk\u00e1 republika: Grada Publishing; 2001: 680.<\/li>\n
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  3. V\u00e1lekov\u00e1 \u013d, Chudej J, Sokol J. Laborat\u00f3rna diagnostika CLL: cytol\u00f3gia, prietokov\u00e1 cytometria. Onkol\u00f3gia.<\/em> 2014;9(3):166\u2013172.<\/li>\n
  4. Balh\u00e1rek T, Barthov\u00e1 M, Sz\u00e9pe P, Marcinek J, Burjanivov\u00e1 T, Plank L. Koment\u00e1r k\u00a0v\u00fdvoju konceptu prognostick\u00fdch faktorov chronickej lymfocytovej leuk\u00e9mie: Cesta od prognostick\u00fdch faktorov k\u00a0prediktorom lie\u010debnej odpovede. Onkol. <\/em>2009;22(6):254\u2013263.<\/li>\n
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  9. Crespo M, Bosh, F, Villamor N, Bellosillo B, Colomer D, et al. ZAP-70 Expression as a\u00a0Surrogate for Immunoglobulin-Variable-Region Mutations in Chronic Lymphocytic Leukemia. N Engl J Med.<\/em> 2003;348(18):1764\u20131775.<\/li>\n
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  14. Urbanov\u00e1 A, Janega P, Mas\u00e1rov\u00e1 K, \u0160tef\u00e1nikov\u00e1 Z, Bab\u00e1l P. Imunohistochemick\u00e1 detekcia prote\u00ednu ZAP-70 a\u00a0jej v\u00fdznam v\u00a0diagnostike B-CLL. \u010cesko-slovensk\u00e1 patologie. <\/em>2009;45(2):40\u201345.<\/li>\n
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  17. Shankey TV, Forman M, Scibelli P, Cobb J, Smith CM, et al. An Optimized Whole Blood Method for Flow Cytometric Measurement of ZAP-70 Protein Expression in Chronic Lymphocytic Leukemia. Cytometry Part B.<\/em> 2006;70B:259\u2013269.<\/li>\n
  18. Bekkema R, Tadema A, Daenen SMGJ, Kluin Nelemans HC, Mulder AB. An Improved Flow Cytometric Method Using FACS Lysing Solution for Measurement of ZAP-70 Expression in B-Cell Chronic Lymhocytic Leukemia. Cytometry Part B.<\/em> 2008;74B:40\u201344.<\/li>\n
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  20. Zucchetto A, Bomben R, Dal Bo M, Nanni P, Bulian P, et al. ZAP-70 Expression in B-Cell Chronic Lymphocytic Leukemia: Evaluation by External (Isotypic) or Internal (T\/NK Cells) Controls and Correlation with IgVH <\/sub>Cytometry Part B.<\/em> 2006;70B:284\u2013292.<\/li>\n
  21. Chen Y, Peterson LC, Dittmann D, Evens A, Rosen S, et al. Comparative Analysis of Flow Cytometric Techniques in Assessment of ZAP-70 Expression in Relation to IgVH <\/sub>Mutational Status in Chronic Lymphocytic Leukemia. Am J Clin Pathol.<\/em> 2007;127:1882\u20131191.<\/li>\n
  22. Best OG, Ibbotson RE, Parker AE, Davis ZA, Orchard JA, Osciier DG. ZAP-70 by Flow Cytometry: A\u00a0Comparison of Different Antibodies, Anticoagulants, and Methods of Analysis. Cytometry Part B.<\/em> 2006;70B:235\u2013241.<\/li>\n
  23. Rossi M, Delprincipe MI, Rossi D, Consalvo MI, Luciano F, et al. Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T\/B ratio versus percentage of positive cells. of Translational Medicine.<\/em> 2010;8:1\u201311.<\/li>\n
  24. Slack GW, Wizniak J, Dabbagh L, Shi X, Gelebart P, et al. Flow Cytometric Detection of ZAP-70 in Chronic Lymphocytic Leukemia.Correlatin With Immunocytochemistry and Western Blot Analysis. Arch Pathol Lab Med. <\/em>2007;131:50\u201356.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    *All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf\u00a0 attachment at the end of the paper.   Chronick\u00e1 lymfocytov\u00e1 leuk\u00e9mia z\u00a0B lymfocytov (B-CLL) je definovan\u00e1 ako n\u00edzkoagres\u00edvne lymfoproliferat\u00edvne ochorenie zapr\u00ed\u010dinen\u00e9 klon\u00e1lnou prolifer\u00e1ciou mal\u00edgne transformovan\u00fdch mal\u00fdch zrel\u00fdch B lymfocytov. Tieto bunky so \u0161pecifick\u00fdm imunofenotypom sa akumuluj\u00fa v\u00a0perif\u00e9rnej<\/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":[291],"tags":[799,798,635,800],"class_list":["post-1196","post","type-post","status-publish","format-standard","hentry","category-imunology","tag-b-cll","tag-flow-based-cytometry","tag-prognostic-markers","tag-zap-70","typ_clanku-review-article"],"acf":{"abstrakt":"

    Chronic lymphocyte leukemia (CLL) is defined as a lymphoproliferative disease caused by clonal proliferation and accumulation of small mature B lymphocytes with malignant transformation in peripheral blood, lymphatic nodes, spleen and bone marrow. Clinical course of CLL is variable. A significant prognostic reach of various CLL biomarkers has been demonstrated, such as IgVH mutation status, chromosomal abnormalities such as CD38 and ZAP-70 expression. Expression of ZAP-70 or CD38 correlates with non-mutated status of IgVH genes and is associated with poorer prognosis. Immunophenotypic analysis by way of flow-based cytometry is suitable not only to detect characteristic immunophenotypic profile of B-cell CLL but also to establish their intracellular expression of ZAP-70.<\/p>\n

    Key words:<\/strong> flow-based cytometry, prognostic markers, B CLL, ZAP-70.<\/p>\n","casopis":[{"ID":1000,"post_author":"7","post_date":"2015-11-21 14:33:24","post_date_gmt":"2015-11-21 13:33:24","post_content":"

    GENETICS<\/strong><\/h4>\r\n
      \r\n \t
    • The laboratory age<\/li>\r\n \t
    • The Odyssey of DNA reading<\/li>\r\n \t
    • Comparative genomic hybridisation: a methodological introduction<\/li>\r\n \t
    • Next generation sequencing and its application in clinical genetics<\/li>\r\n<\/ul>\r\n

      <\/h4>\r\n

      BIOCHEMISTRY<\/strong><\/h4>\r\n
        \r\n \t
      • Determining of the trace elements in blood serum<\/li>\r\n \t
      • Determining of \u03b1-tocopherol (vitamin E) in serum by way of liquid chromatography with tandem mass spectrometry (LC\/MS\/MS)<\/li>\r\n \t
      • Analysis of urinary calculi and its path to Europe<\/li>\r\n<\/ul>\r\n \r\n

        IMMUNOLOGY <\/strong><\/h4>\r\n
          \r\n \t
        • New autoantibodies in diagnosis of autoimmunity myopathies<\/li>\r\n \t
        • Taking advantage of flow-based cytometry in determining prognostic markers<\/li>\r\n<\/ul>\r\nCD38 and ZAP-70 in patients with B-CLL\r\n\r\n \r\n

          CYTOLOGY AND PATHOLOGY <\/strong><\/h4>\r\n
            \r\n \t
          • Mucinous ovarian carcinoma \u2013 news in diagnosis from the pathologist\u2019s perspective<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-12015","to_ping":"","pinged":"","post_modified":"2017-08-16 21:43:10","post_modified_gmt":"2017-08-16 19:43:10","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/casopis\/newslab-12015\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"39","upload_clanok":{"ID":1197,"id":1197,"title":"medirex_1_2015-elbertova","filename":"MEDIREX_1_2015-\u2013-Elbertova.pdf","filesize":131611,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2016\/12\/MEDIREX_1_2015-\u2013-Elbertova.pdf","link":"https:\/\/www.newslab.sk\/en\/application-of-flow-based-cytometry-in-establishing-prognostic-cd38-and-zap-70-markers-in-patients-with-b-cll\/medirex_1_2015-elbertova\/","alt":"","author":"7","description":"","caption":"","name":"medirex_1_2015-elbertova","status":"inherit","uploaded_to":1196,"date":"2016-12-06 21:21:32","modified":"2016-12-06 21:21:32","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1196","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1196"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1196\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1196"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1196"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1196"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}