Hemato Flow Novi pristup u analizi DKS-a
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1 Hemato Flow Novi pristup u analizi DKS-a Anica Remenar Beckman Coulter
2 Uzorak pune krvi Hematološki analizator DA Oznake?? Poruke?? NE Razmaz Validacija
3 Abnormalni uzorci Brojanje 100 stanica pod mikroskopom Pojavom sumnjivih stanica razmaz se šalje citologu 100 CELLS COUNT AT MICROSCOPE: 1 ST RUN 3 EO, 0 MYELOCYTES 2 ND RUN 1 EO, 4 MYELOCYTES
4 Ograničenja ručne analize diferencijalne kvne slike Pogrešan izbor područja brojenja na razmazu Rezultat ovisi isključivo o osobi koja mikroskopira Slaba učinkovitost (zahtijeva puno vremena) Statističke pogreške u brojanju
5 Rümke Table
6 Automatizacija : Hemato-Flow or Analyser LH 750 (or DxH800) REMISOL FC 500 Autre Cytodiff Sample Prep FP 1000
7 Uzorak pune krvi Hematološki analizator DA Oznake?? Poruke?? Hematoflow Hematoflow oznake/poruke DA Razmaz NE Validacija
8 Hemato-Flow Cyto Diff panel 1 bočica 6 monoklonskih antitijela 5 boja 16 populacija CD16PC5 : zreli i nezreli granulociti, citotoksični limfociti, limfociti T/NK CD2+CD294+PE : eozinofili, bazofili, aktivirani T, T i NK stanice CD45PC7 : leukociti CD36FITC : monociti CD19ECD : limfociti B i B prekursori.
9 Abnormalni uzorci Analiza 20,000 stanica Analiza stanica protočnim citometrom (1) CytoDiff
10 Protočna citometrija Automatizirana fluorescentna mikroskopija grupa analitičkih metoda koje omogućavaju multiparametarsku analizu morfoloških, biokemijskih i funkcionalnih karakteristika stanica, staničnih organela i unutarstaničnih molekula Ciljevi automatizacije: analiza pojedinačnih stanica analiza uzorka relevantne veličine (nekoliko desetaka tisuća čestica uzorka) analiza u razumnom vremenu (>10 3 stanica/s) objektivna metoda koja isključuje subjektivnu procjenu fluorescencije pod mikroskopom
11 Stanični fenotip na membrani svake stanice eksprimirane su molekule (proteini i glikoproteini) različitih bioloških funkcija koje nazivamo STANIČNIM DIFERENCIJACIJSKIM ANTIGENIMA vrsta i broj odreďenih diferencijacijskih antigena na stanicama naziva se fenotipom
12 Princip protočne citometrije Side Scatter (SSC) Stupanj raspršenja svjetlosti pod pravim kutom od laserske zrake ~ Struktura stanice Forward Scatter (FSC) Stupanj raspršenja svjetlosti u smjeru laserske zrake pod malim kutom Laser ~ Veličina stanice Fluorescence Intensity Y
13 OdreĎivanje staničnog fenotipa površinski stanični antigeni fluorokromima obilježena monoklonska antitijela Y
14 Primjena protočne citometrije u biomedicini Imunofenotipizacija stanica (odreďivanje staničnih diferencijacijskih antigena pomoću fluorokromima obilježenih monoklonskih antitijela) OdreĎivanje unutarstanične DNA/RNA (važna komponenta dijagnostike malignih bolesti) Analiza funkcionalnih obilježja stanice (detekcija unutarstaničnih citokina, enzima, iona) Analiza bakterija, gljiva, algi
15 Imunofenotipizacija vrsta stanice stupanj sazrijevanja stanice vijabilnost stanice i podatak o tipu stanične smrti da li je stanica maligno transformirana ili nije da li je aktivirana, da li proliferira
16 Cyto Diff 16 parametara diff 9 CE parametara: 1. B Limfociti 2. T/NK Limfociti 3. Ukupni Limfociti 4. Ukupni Monociti 5. Neutrofilni granulociti 6. Eozinofili 7. Bazofili 8. Nezreli Granulociti 9. Blasti ukupno
17 Cyto Diff 16 parametara diff 7 RUO parametara 1. Nezreli T limfociti 2. Nezreli B limfociti 3. Nezreli non-t i non-b leukociti 4. Granulirani T i NK stanice 5. Ne citotoksični-t i NK stanice 6. Upalni monociti 7. Ne upalni monociti
18 Postupak PrepPlus2 - Brza priprema uzorka: krv + CytoDiff cca min za pripremu 12 uzoraka (ili ručno pipetiranje) 10 min inkubacija (liza se pokreće automatski na TQ Prep Workstation nakon ovih 10 min inkubacije) TQ Prep Workstation - liza eritrocita: non wash metoda - VersaLyse cca 10 min za 12 uzoraka Analiza: WBC cca 1 min po uzorku ovisno o broju WBC u uzorku
19 HematoFlow više nije koncept nego realnost Diferencijalna krvna slika (DKS) pomoću hematološkog brojača i mikroskopski pregled razmaza ograničavaju nas na samo 5 leukocitinih subpopulacija Identifikacija abnormalnih stanica je samo kvalitativna, često problematična sa slabo ponovljivim rezultatima (analiza samo 100 stanica)
20 HematoFlow Nova metoda odreďivanja diferencijalne krvne slike metodom protočne citometrije sa 6 markera, koristeći kombinaciju od 5 fluorescentnih boja; CD36-FITC/CD2- PE1CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 protočna citometrija omogućava istodobno mjerenje nekoliko strukturalnih i funkcionalnih parametara svake pojedinačne stanice u uzorku
21 100 Uzoraka pune krvi Hematološki analizator DA Oznake?? Poruke?? 20 Hematoflow Hematoflow oznake/poruke DA 3 Razmaz NE 17 Validacija
22 Prednosti automatizirane analize DKS-a Prednost analize diferencijalne krvne slike protočnom citometrijom je u brzoj analizi velikog broja leukocita (obično leukocita) Analiza protočnom citometrijom bolja je u detekciji i kvantifikaciji blastičnih stanica i/ili nezrelih granulocita TakoĎer, prednost analize DKS-a protočnom citometrijom je u detekciji CD16 pozitivnih upalnih monocita
23 Cytometry Part A 77A: , hematoloških uzoraka (1 dan) - normalno 25 % treba ručno diferenciranje ( slideova dnevno) Nakon CytoDiff samo 22 uzorka trebala ručni diff (+ 16 koji su imali RBC flags)
24 Iz zaključka: WBC differential by FCM is at least as reliable as by standard cytology FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case
25 Zaključak Istraživanja1,2 pokazuju da je kvantitativna detekcija abnormalnosti diferencijalne krvne slike neutrofila, eozinofila, bazofila, monocita i limfocita metodom protočne citometrije jednako pouzdana kao i standardna mikroskopija krvnih razmaza Cyto Diff je bolji od citologije u detekciji nezrelih granulocita i blasta 1. Jean-Luc Faucher et all, 6 Markers/5 Colors Extended White Blood Cell Differential by Flow Cytometry, Cytometry Part A, 71A: 934_944, Sven Bjo rnsson, Total Nucleated Cell Differential for Blood and Bone Marrow Using a Single Tube in a Five-Color Flow Cytometer, Cytometry Part B (Clinical Cytometry) 74B: (2008)
26 Thank you for your attention
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