1. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Immunophenotyping and applications of cell analysis in the hematology laboratory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering April 5, 2005 This lecture can be found on http://www.cyto.purdue.edu/class

2. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor What can flow cytometry be used for? Immunology Hematology Pathology Microbiology Genetics Drug discovery Toxicity testing Cell culture studies Functional studies Clinical and Research Chemical Engineering Biotechnology Agronomy Animal Sciences

3. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor CELLULAR ANTIGENS Adhesion Receptors Metabolic cytokines structure enzymes Slide courtesy of Jim Bender T cells B Cells Phenotype: …outward physical manifestation…

4. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Cluster Designations (CD) These are based on the Immunology Workshop an international committee that meets in Boston every few years Each antigen that is defined on cells is given a unique number Until a final number is agreed, antigens can be designated CDw (w=workshop a tentative designation) Here is an example of the possible CDspossible CDs

5. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Immunofluorescence staining specific binding nonspecific binding Slide from Dr. Carleton Stewart

6. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Direct staining Fluorescent probe attached to antibody Specific signal: weak, 3dyes/site Nonspecific binding: low Slide from Dr. Carleton Stewart

7. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Avidin-Biotin method I biotinylated primary Ab biotin avidin biotinylated dye

8. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor CFU-GM MYELOBLAST MYELOCYTE META- MYELOCYTE BAND PMN CD16 CD11b CDw13 CD33 CD34 HLA-Dr CD38 CD71 MY8 Myelomonocytic Antigen Distribution Purdue Cytometry Labs PROGRANULOCYTE

9. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Negative Positive Decision Tree in Acute Leukemia HLA-DR T CD13,33 CD19 TdT CD10 CD20 Mu B,T AMLL AML T-ALL AML-M3 AUL ? PRE-BI PRE-BII PRE-BIII PRE-BIVPRE-BV CD13,33 From Duque et al, Clin.Immunol.News.

10. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor What are the principles in flow cytometry? Light scattered by a laser or arc lamp Specific fluorescence detection Hydrodynamically focused stream of particles Electrostatic particle separation for sorting Multivariate data analysis capability

11. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Concepts Scatter: Size, shape, granularity, polarized scatter (birefringence), structure Fluorescence: Intrinsic: Endogenous pyridines and flavins Extrinsic: All other fluorescence profiles Absorption: Loss of light (blocked) Time: Useful for kinetics, QC Count: Number of cells collected in a histogram

12. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Clinical Analyzers

13. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Cell Sorters (FACS – Fluorescence Activated Cell Sorter)

14. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample

15. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Signal direction Flow Chamber

16. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor

18. Light Scatter Materials scatter light at wavelengths at which they do not absorb If we consider the visible spectrum to be 350-850 nm then small particles (< 1/10 ) scatter rather than absorb light For small particles (molecular up to sub micron) the Rayleigh scatter intensity at 0 o and 180 o are about the same For larger particles (i.e. size from 1/4 to tens of wavelengths) larger amounts of scatter occur in the forward not the side scatter direction - this is called Mie Scatter (after Gustav Mie) - thus forward scatter is related to size (at 1-15 microns) Shapiro p 79

19. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Optics for forward scatter scatter detector iris blocker Laser beam Stream in air or a round capillary

20. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Frequency distribution Number of events Intensity of parameter (e.g. fluorescence) histogram

21. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor histogram Intensity of parameter Number of events

22. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Flow cytometry measurements L M G SCATTER FLUORESCENCEIMAGE

23. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor 0 200 400 600 8001000 Side Scatter Projection Light Scatter Gating Forward Scatter Projection 90 Degree Scatter Neutrophils Lymphocytes Monocytes Forward Scatter Human white blood cells

24. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Different size cells Particle or cell size (log scale) Number of events small large 0.1 1 10 100 1000 0.9 20 90 200 700 While forward light scatter is not always related to cell size, in The majority of cases between 1-20 microns, it is a reasonable estimate

25. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Light Scatter of white blood cells Light scatter can be used to identify populations of cells x In peripheral blood, the three main populations of leukocytes can be distinguished. A “gate” or “bitmap” can be placed around a region so that further analysis can be made on this region. The cells in the region marked “X” can be evaluated as a population.

26. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Fluorescence - e.g. Monoclonal Antibodies

27. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor “B” Cells “T” Cells

28. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor 3 Parameter Data Display Isometric Display

29. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor The Cell Cycle G1G1 M G2G2 S G0G0 Quiescent cells

30. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Definitions & Terms Ploidy –related to the number of chromosomes in a cell Haploid: Number of chromosomes in a gamete (germ cell) is called the HAPLOID number for that particular species Diploid: The number of cells in a somatic cell for a particular species Hyperdiploid: greater than the normal 2n number of chromosomes Hypodiploid: Less than the normal 2n number of chromosomes DNA Tetraploidy: Containing double the number of chromosomes DNA Index: The ratio between the mode of the relative DNA content of the test cells (in G 0 /G 1 phase) to the mode of the relative DNA content in normal G 0 /G 1 diploid cells Coefficient of Variation - CV: The ratio between the SD of the mode of the G 0 /G 1 cell populations expressed as a percentage.

31. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Normal Cell Cycle G0G0G0G0 G0G0 - G 1 s G2G2 M DNA Content 2N 4N G2G2 M G0G0 G1G1 s 0 200 400 600 8001000 0 75 150 225 300 Cell Count

32. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor A typical DNA Histogram G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events 2n 4n

33. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Flow Cytometry of Apoptotic Cells PI - Fluorescence # Events Apoptotic cells Normal G0/G1 cells

34. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Analyzing the DNA Histogram

35. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Chromosome Analysis Most human chromosomes can be separated by flow cytometry

36. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Chromosome Analysis (BivariateFlow Karyotyping - porcine) (Bivariate Flow Karyotyping - porcine) chromosome 1 chromosome 2

37. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Ethidium PI 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Spectra of PI and EtBr

38. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor log Thiazole Orange.1 1000 100 10 1 Count 0 150 112 75 37 RMI = 0 log Thiazole Orange.1 1000 100 10 1 Count 0 150 112 75 37 RMI = 34 Reticulocyte Analysis

39. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor 4 colors - simultaneous collection (can go to 17 colors) Emission wavelength (nm) 530 580630 680 730 780 FITC PE PE- TR PE-CY5 We separate different subsets by taking bands of light from the light spectrum and analyzing the intensity of light in that band

40. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor CD3 CD56 CD8 FOUR COLOR PATTERN CD4CD8 CD56 - NK CD8CD4 Data from Dr. Carleton Stewart CD56 – NK Cells CD3 – T cells CD4 – T cells – Helper CD8 – T cells - Cytotoxic This is a subset of cells It is CD3 + CD56 + This is a subset of cells It is CD3 + CD4 +

41. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Multicolor Analysis Roederer, et al

42. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor

43. Cellular Response: Cell death Cell death Cell ‘suicide’ Cell ‘suicide’ Ignore damage Ignore damage Damage repair Damage repair Incorrect repair Incorrect repair

44. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Functional Assays intracellular pH intracellular calcium intracellular glutathione oxidative burst phagocytosis

45. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Oxidative Burst generation of toxic oxygen species by phagocytic cells superoxide anion measured with hydroethidine hydrogen peroxide measured with 2’,7’-dichlorofluorescin diacetate (DCFH-DA)

46. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor TIME (seconds) 0 2400 1800 1200 600 Scale 345 115 38 12 4 Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Unstimulated Neutrophils

47. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor FITC-Labeled Bacteria Phagocytosis

48. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Cellular Functions Cell Viability Phagocytosis Organelle Function –mitochondria, ER –endosomes, Golgi Oxidative Reactions –Superoxide –Hydrogen Peroxide –Nitric Oxide –Glutathione levels Ionic Flux Determinations –Calcium –Intracellular pH Membrane Potential Membrane Polarization Lipid Peroxidation

49. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Organelle Function MitochondriaRhodamine 123 EndosomesCeramides GolgiBODIPY-Ceramide Endoplasmic ReticulumDiOC 6 (3) Carbocyanine

50. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Fluorescent Indicators How the assays work: Superoxide: Utilizes hydroethidine the sodium borohydride reduced derivative of EB Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H 2 O 2 ) oxidize the DCFH to fluorescent DCF Glutathione: In human samples measured using 40  M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time. Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar manner to H 2 O 2 so care must be used. DAF is a specific probe available for Nitric Oxide

51. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Hydroethidine HE EB N CH 2 CH 3 NH 2 H2NH2N H Br - N CH 2 CH 3 NH 2 H2NH2N + O2-O2- Phagocytic Vacuole SOD H2O2H2O2 NADPH NADP O2O2 NADPH Oxidase OH - O2-O2- DCF HE O2-O2-O2-O2- H2O2H2O2H2O2H2O2 DCF Example: Neutrophil Oxidative Burst

52. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H2O2H2O2 DCFH-DADCFH-DA DCFH DCF H O 2 2 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence.1.1 1000 100 10 1 0 20 40 60 counts PMA-stimulated PMN Control 80

53. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Phagosome O2O2 O2-O2- H2O2H2O2 NADPH + H + NADP + HMP NADPH Oxidase GSSG GSH GR GP H2O2H2O2 SOD O2-O2- H+H+ H2OH2O Catalase H 2 O + O 2 PCB SOD PCB (Reduced GSH level) Stimulant PKC PCB (PMA) Human Neutrophil ? ? + O2-O2- OH. Lipid Peroxidation Phospolipase A2 activity Leukotrienes H2O2H2O2

54. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Hydroethidine Superoxide Production 15 minutes45 minutes

55. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Cell Sorting Physically separating cells based on some measurable characteristic Placing these cells into containers

56. 488 nm laser + - Fluorescence Activated Cell Sorting Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector Purdue University Cytometry Laboratories

57. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor

58. Cell Sorting Video of the droplet formation in a sort stream from a Cytomation instrument. Source: Purdue CDROM vol 4, 1998 Video2.mpg +++ ---

59. Purdue University Cytometry Laboratories – J. Paul Robinson, Professor Lab 2 Groups of 8 students each Hansen Hall, Room B50 (Basement) Meet with Kathy Ragheb and Cheryl Holdman One on April 18, 12:30-4:30 Other April 25, 12:30-4:30 Work in groups of 2 and you will take blood Phenotype your own blood for T cell and B cells Blood film and total blood count Coulter count to obtain total cell numbers Look at T and B cells under fluorescence scope