Whole Blood Flow Cytometry

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1 Whole Blood Flow Cytometry y Nailin Li Department t of Medicine, i Clinical i l Pharmacology Unit Karolinska Institute/University Hospital, Stockholm Department of Pathology & Pathophysiology Zhejiang University School of Medicine Hangzhou

Institute/University Hospital, 171 76 Stockholm Department of

2 QuickTime?and a GIF decompressor are needed to see this picture. BMJ 2003; 326:354

3 Neutrophil Eosinophil Erythrocyte t Monocyte Platelets Basophil Lymphocyte

4 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

5 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

6 Blood Sampling - I No stasis or with stasis? - no stasis or light stasis for platelet samples What needle to be used? - wide calibre puncture needles ( 21 gauge) better Venepuncture - clean venepuncture for monocytes and platelets l t - avoid the first 2 ml blood

7 Blood Sampling - II Pre-staining sample handling - duration before staining platelets: 10 min monocytes/granulocytes: 30 min lymphocytes: hours to days - storage temperature room temperature 4 C maintain consistent storage temperature!!! - sample fixation recommendable for delayed staining

hours to days - storage temperature room temperature 4 C maintain

8 Anticoagulants Recommanded Sodium citrate Acid-citrate-dextrose (ACD) Hirudin and its analogues Not recommanded Heparin EDTA

9 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

10 Flow cytometric dot plot of a whole blood sample leukocytes erythrocytes t platelets

11 Approaches for target t cell isolation Gating on light scattering signalings Hemolytic target cell isolation Identification by fluorescent labelling Cell purification by magnetic beads

12 Platelet identification from other blood cells by light scattering signals platelets

13 Leukocyte isolation by hemolysis - Ammonium chloride lysing solution - Basis of automation of sample preparation QuickTime?and a TIFF (LZW) decompressor are needed to see this picture. BD FACS Lyse/Wash Assistant Coulter TQ-Prep Workstation

14 Hemolysis and washing may cause artefacts A B E h L i Prefixation Erythrocyte Lysis PLA A/L % * * PLA A/L % * Unfixed F-I F-II Unlysed Lysed * P<0.05 Li N, et al. Br J Haematol 1997;99:808.

A/L % 80 60 40 * * PLA A/L % 80 60 40 * 20 20 0 0

15 Whole blood flow cytometry Li N, et al. Br J Haematol 1997;99:808.

16 Whole blood flow cytometry with a fluorescence triggering Li N, et al. Eur J Hemotol 2000; 65: 57.

17 Precise detection of platelet-leukocyte aggregation by WB FCM with fluorescence triggering 50 PLA (%) w/o blocking cocktail with blocking cocktail * P < * 0 Unstimulated ADP 10 µm Li N, et al. Cytometry 1999;35: 154.

blocking cocktail with blocking cocktail * P < 0.

18 Leukocyte isolation by magnetic beads Dynal beads QuickTime?and a TIFF (LZW) decompressor are needed to see this picture. QuickTime?and a TIFF (LZW) decompressor are needed to see this picture. - positive isolation - negative isolation

19 Whole blood flow cytometry with fluorescent signal triggering is the choice for sensitive blood cells!!

20 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

21 Triggering fluorescent marker for WB FCM: principle ot the selection Antigen: surface antigen constitutive expression high expression density Fluorochrome: the brighter the better R-phycoerythrin (PE) Choice for leukocytes: PE-CD45

22 Scheme of classical flow cytometry for blood samples Blood samples Fluorescent staining Hemolysis Dilution and fixation Flow cytometric analysis

23 Scheme of classical flow cytometry for blood samples Blood samples Fluorescent staining Hemolysis Dilution and fixation Flow cytometric analysis

24 Scheme of whole blood flow cytometry Blood samples Fluorescent staining Dilution and fixation Flow cytometric analysis

25 Sample incubation Early sample handling better, eg, <10 min of blood collection Incubate sample with 1:10 dilution and avoid stirring/agitation to reduce cell conjugation/aggregation Incubation with saturating ti concentrations ti of MAbs for cell identifation and/or activation markers Agonist added to test cell reactivity in vitro Incubation at 22 C for for min in dark

26 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

27 Sample storage Light fixation recommendable Temperature 22 C FCM analysis < 0.5-1hr 4 C delayed analysis Sample stability very individual, depending on cell types, antigen, and storage conditions

28 Flow cytometry t for blood cell analysis blood collection and anticoagulation isolation/identification of target cells fluorescent labelling of target cells sample storage and stability flow cytometric analysis

29 What can we do with whole blood flow cytometry?

30 WB FCM application: cell phenotyping Count pan leukocytes all T cells Tc cells RPE-CD45 PC5-CD3 ECD-CD8 FITC-C -CD42a Plt-Tc aggr. Forward Scattering large cells small cells ECD-CD8 Side Scattering Li N, et al. J Thromb Haemost2006;4:874.

31 WB FCM application: cell phenotyping Li N, et al. Cytometry 1999;35:154.

32 WB FCM application: detection of cell activation Li N, et al. Eur J Hemotol 2000;65:57.

33 WB FCM application: detection of cell activation RBC+WBC Unstimulated QuickTime?and a TIFF decompressor are needed to see this picture. Platelets P-selectin expression positive 10 µm ADP QuickTime?and a TIFF decompressor are needed to see this picture. Activation-related surface antigen changes in platelets

34 WB FCM application: Ca 2+ signaling g QuickTime?and a TIFF decompressor are needed to see this picture. 0.1 U/ml thrombin mm CaCl 2 Thrombin induced change of platelet cytosolic Ca 2+ Thrombin-induced change of platelet cytosolic Ca 2+ (fluorescent Ca 2+ indicators: Fluo-3, Indo-1, and Fura-2)

35 WB FCM application: Ca 2+ signaling g luo-3 MFI F µm ADP With [Ca 2+ ] E 0 Without [Ca 2+ ] E Time (s) ADP-induced platelet cytosolic Ca 2+ increase in the absence or presence of extracellular Ca 2+

36 WB FCM application: blood cell counting platelet aggregates QuickTime?and a TIFF decompressor are needed to see this picture. single platelets fluorecent beads microparticles Flow cytometric platelet counting assay Flow cytometric platelet counting assay using fluorescence beads

37 WB FCM application: platelet shape change QuickTime?and a TIFF decompressor are needed to see this picture. Unstimulated M ADP Shape change rate(%) = (D S A 0 D 0 A S )/D 0 A S 100% A 0, D 0, A S and D S stand for the numbers of events in bit maps A and D in unstimulated and stimulated samples, respectively

38 Platelets

39 WB FCM application: reticulated platelet analysis Use fluorescent nucleic acid dyes, e.g, thiazole orange (TO), to identify newly-released, RNA-positive platelets. QuickTime?and a TIFF decompressor are needed to see this picture. Harrison P, et al. Platelets 1997;8:379.

40 Advantages of whole blood flow cytometry y Blood cell function studied in the physiological milieu of whole blood. Minimal sample manipulation minimizes artificial cell activation in vitro, and prevents potential selective loss of blood cell subpopulations. Both blood cell activation in vivo and in vitro can be mornitored. High assay efficiency.

41 Keypoints of whole blood FCM Clean venepuncture with citrate or hirudin as the anticoagulant. Immediate ( 10 min) sample preparation with no cell separation or washing/centrifugation if ti processes. Sample incubation: 2-5 µl blood, 1:10 dilution, 22 C, no stirring, in dark, min. If appliable, mildly fix samples with 0.2-1% formaldehyde or 0.5-2% paraformaldehyde. Store samples at 4 C for delayed danalysis. Standardize the interval between sample prepara- Standardize the interval between sample prepara tion and analysis.

42 Whole blood flow cytometry with a fluorescence triggering 1000 Histogram 3 Count 28 0 Histogram 1 Pan Leukocytes Fo orward Scat ttering RPE-CD Lym Histogram 2 ITC-GPIX F unt.1 28 P-Neu Neus RPE-CD45 Mon Neu Histogram 4 Co Side Scattering Neu FITC-CD11b CD11b Hu H, et al. Thromb Haemost 2003;90:679.

No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer

APPLICATION NOTE Attune NxT Flow Cytometer No-wash, no-lyse detection of leukocytes in human whole blood on the Attune NxT Flow Cytometer Introduction Standard methods for isolating and detecting leukocytes