Immunophenotyping and applications of cell analysis in the hematology laboratory

1. Immunophenotyping and applications of cell analysis in the hematology laboratory J.Paul Robinson Professor of Immunopharmacology & Biomedical Engineering April 5, 2005

2. What can flow cytometry be used for? Clinical and Research • Immunology • Hematology • Pathology • Microbiology • Genetics • Drug discovery • Toxicity testing • Cell culture studies • Functional studies • Chemical Engineering • Biotechnology • Agronomy • Animal Sciences

3. Phenotype: …outward physical manifestation… CELLULAR ANTIGENS cytokines structure enzymes Adhesion Receptors Metabolic T cells B Cells Slide courtesy of Jim Bender

4. 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 CDs

5. Immunofluorescence staining specific binding nonspecific binding Slide from Dr. Carleton Stewart

6. Direct staining • Fluorescent probe attached to antibody • Specific signal: weak, 3dyes/site • Nonspecific binding: low Slide from Dr. Carleton Stewart

7. Avidin-Biotin method I biotinylated primary Ab biotin avidin biotinylated dye

8. Myelomonocytic Antigen Distribution CDw13 MY8 CD11b CFU-GM CD16 PROGRANULOCYTE MYELOCYTE META- BAND MYELOCYTE MYELOBLAST PMN HLA-Dr CD34 CD33 CD38 CD71 Purdue Cytometry Labs

9. From Duque et al, Clin.Immunol.News. PRE-BV PRE-BIV Negative Positive Mu PRE-BIII PRE-BII CD20 AUL PRE-BI CD10 TdT AMLL AML ? AML-M3 CD19 B,T CD13,33 T-ALL CD13,33 T HLA-DR Decision Tree in Acute Leukemia

10. What are the principles in flow cytometry? • Lightscattered by a laser or arc lamp • Specific fluorescence detection • Hydrodynamically focused stream of particles • Electrostatic particle separation for sorting • Multivariate data analysis capability

11. 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. Clinical Analyzers

13. Cell Sorters (FACS – Fluorescence Activated Cell Sorter)

14. Optical Design PMT 5 PMT 4 Sample PMT 3 Dichroic Filters Flow cell PMT 2 Scatter PMT 1 Laser Sensor Bandpass Filters

15. Signal direction Hydrodynamic Systems Sample in Sheath Piezoelectric crystal oscillator Sheath in Fluorescence Sensors Laser beam Scatter Sensor Sheath Core Flow Chamber

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 0o and 180o 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. Optics for forward scatter iris Laser beam scatter detector blocker Stream in air or a round capillary

20. Number of events Intensity of parameter (e.g. fluorescence) Frequency distribution histogram

21. histogram Number of events Intensity of parameter

22. Flow cytometry measurements SCATTER FLUORESCENCE IMAGE G M L

23. Light Scatter Gating Side Scatter Projection Neutrophils Forward Scatter Forward Scatter Projection Monocytes Lymphocytes 200 400 600 800 1000 0 90 Degree Scatter Human white blood cells

24. Different size cells 700 200 90 20 Number of events 0.9 small large 0.1 1 10 100 1000 Particle or cell size (log scale) 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. Light Scatter of white blood cells • Light scatter can be used to identify populations of cells 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. x

26. Fluorescence - e.g. Monoclonal Antibodies

27. “T” Cells “B” Cells

28. 3 Parameter Data Display Isometric Display

29. The Cell Cycle M G2 G1 Quiescent cells G0 S

30. 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 G0/G1phase) to the mode of the relative DNA content in normal G0/G1 diploid cells • Coefficient of Variation - CV: The ratio between the SD of the mode of the G0/G1 cell populations expressed as a percentage.

31. 300 225 150 G2 M 75 0 0 200 400 600 800 1000 Normal Cell Cycle G0 - G1 G0 G0 M G2 G1 s Cell Count s 2N 4N DNA Content

32. A typical DNA Histogram 4n 2n G0-G1 G2-M S # of Events Fluorescence Intensity

33. Apoptotic cells Normal G0/G1 cells # Events PI - Fluorescence Flow Cytometry of Apoptotic Cells

34. Analyzing the DNA Histogram

35. Chromosome Analysis Most human chromosomes can be separated by flow cytometry

36. Chromosome Analysis (BivariateFlow Karyotyping - porcine) chromosome 1 chromosome 2

37. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm PI Ethidium Spectra of PI and EtBr

38. Reticulocyte Analysis 150 150 112 112 RMI = 34 RMI = 0 Count Count 75 75 37 37 0 0 1000 100 .1 1 1000 1 10 10 100 .1 log Thiazole Orange log Thiazole Orange

39. 4 colors - simultaneous collection(can go to 17 colors) FITC PE PE-CY5 PE- TR 530 580 630 680 730 780 Emission wavelength (nm) We separate different subsets by taking bands of light from the light spectrum and analyzing the intensity of light in that band

40. FOUR COLOR PATTERN 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+ CD8 CD4 CD56 - NK CD3 CD3 CD3 CD8 CD4 CD4 CD56 CD56 CD8 Data from Dr. Carleton Stewart

41. Multicolor Analysis Roederer, et al

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

44. Functional Assays • intracellular pH • intracellular calcium • intracellular glutathione • oxidative burst • phagocytosis

45. 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. Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Scale Unstimulated Neutrophils 345 115 38 12 4 2400 0 600 1200 1800 TIME (seconds)

47. Phagocytosis FITC-Labeled Bacteria

48. Cellular Functions • Ionic Flux Determinations • Calcium • Intracellular pH • Membrane Potential • Membrane Polarization • Lipid Peroxidation • Cell Viability • Phagocytosis • Organelle Function • mitochondria, ER • endosomes, Golgi • Oxidative Reactions • Superoxide • Hydrogen Peroxide • Nitric Oxide • Glutathione levels

49. Organelle Function • Mitochondria Rhodamine 123 • Endosomes Ceramides • Golgi BODIPY-Ceramide • Endoplasmic Reticulum DiOC6(3) Carbocyanine

50. 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 (H2O2) 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 H2O2 so care must be used. DAF is a specific probe available for Nitric Oxide