1. Lecture 2 Clinical Applications of Flow Cytometry
This lecture will discuss the current applications of flow cytometry in the clinical environment. Many of these methods are not yet applicable in veterinary medicine because of cost and development of specific monoclonal antibodies. However, there are now developments on the horizon that will reduce the cost and size of flow cytometers which will influence the use of these tools in clinical veterinary medicine. This lecture will outline the possible applications and the advantages they might have over competing technologies.

2. Clinical Applications of Flow Cytometry
J.Paul Robinson
Professor of Immunopharmacology
Professor of Biomedical Engineering
Purdue University
School of Veterinary Medicine

3. Primary areas
DNA/RNA Analysis
Microbiology
Phenotyping
Cell Function

4. Purdue University Cancer Center & Purdue University Cytometry Laboratories

5. Brief Introduction to Flow Cytometry
What do these instruments look like?
What does flow cytometry do?
How does it work?
Why is it useful?

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

10. A Histogram (a frequency distribution graph)
# of Events
Increase in Fluorescence Intensity 17

11. DNA Probes DNA in cells can be stained with a fluorescent dye
DNA probes like Propidium Iodide are STOICHIOMETRIC – that means the number of molecules of probe bound is equivalent to number of molecules of DNA
So we can measure how much DNA is in a cell

12. DNA/RNA Probes Propidium Iodide Hoechst Cyanine Dyes Acridine Orange
TOTO-1 , YOYO-1, TOTO-3
Thiazole Orange, Thiazole Blue, Thioflavin
PRO dyes
SYTO/SYTOX dyes (Sytox green)
Acridine Orange
Pyronin Y
Styryl Dyes
Mithramycin + EtBr

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

14. 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

15. Definitions & Terms
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

16. Definitions & Terms
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.

17. A DNA histogram
G0-G1
Cell Number
G2-M S
Fluorescence Intensity 14

18. A typical DNA Histogram
G0-G1
G2-M S
# of Events
Fluorescence Intensity 17

19. Multiparameter gating
R1-gate
Cyclin - B1 - FITC
Mitotic cells
P-105 Cy5
Endoduplicating population
DNA - Hoechst
DNA - Hoechst
Human Prostate tumor cell line DU-145
Data from Dr. James Jacobberger

20. DNA Analysis DNA Analysis 2N 4N Aneuploid peak PI Fluorescence
200
400
600
800
1000
PI Fluorescence
DNA Analysis 2N 4N
DNA Analysis
Aneuploid peak
200
400
600
800
1000
PI Fluorescence

21. Reticulocyte Analysis
RMI = 34
RMI = 0
use of maturity index decreases interlab variability
RMI = fraction of highly fluorescent retics/total retics
cursor is set to exclude nucleated cells .1 1 10
100
1000 .1 1 10
100
1000
log Thiazole Orange
log Thiazole Orange

22. Reticulocyte Analysis
log Thiazole Orange .1
1000
100 10 1 R1 R2 R3 R4
RMI = 34

23. Measurement of Apoptosis
Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”.
Characteristics are condensation of the chromatin material
Blebbing of nuclear material
Often accompanied by internucleosomal degradation of DNA giving rise to distinctive 'ladder' pattern on DNA gel electrophoresis.

24. Detection Methods for Apoptotis
Phosphatidyl serine, can be detetected by incubating the cells with fluorescein-labeled Annexin V
By staining with the dye, Hoechst (UV)
By staining with the dye PI (visible)
By staining with the dye YOPRO-1 (visible)

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

26. Labeling Strand Breaks with dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)]
Green:
apoptotic cells
R2: Apoptotic Cells
Green Fluorescence
Side Scatter
R1:
Normal
Cells
Red:
normal cells
PI-Red Fluorescence
Forward Scatter
Green Fluorescence
Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidin
Red fluorescence is DNA counter-stained with 20µg/ml PI

27. Nuclear Antigens Ki-67 - proliferation related antigen
Ki-S1 - proliferation related antigen
Cyclin A: expression begins in late G1/early S phase and increases as cells traverse S phase, reaching a maximum in G2. Cyclin A is not expressed in mitotic cells
Cyclin B1: accumulates in late S phase but is maximally expressed in G2 and mitosis.

28. Nuclear antigens Human Prostate tumor cell line DU-145 DNA - Hoechst
P-105 -CY5
DNA - Hoechst
Cyclin - B1 - FITC (log)
FALS
90 deg Scatter (log)
Cyclin - B1 - FITC
Human Prostate tumor cell line DU-145
Data from Dr. James Jacobberger

29. Differential Inflammatory Cell Count
Data from Dr. Doug Redelman, Sierra Cytometry

30. Simultaneous UV & Visible Light
PI only binds to DNA where it can gain access to the cell - ie Dead cells
Hoechst binds to
all DNA - It is UV excited
PI - fluorescence
Hoechst (UV)
Hoechst
Data from Dr. Doug Redelman, Sierra Cytometry

31. Hoechst & PI Fluorescence
Data from Dr. Doug Redelman, Sierra Cytometry

32. Boar Sperm Hoechst/PI Dead FL2-PI FL1-Hoechst
Data from Dr. Doug Redelman, Sierra Cytometry

33. Human Sperm Sybr green PI
Data from Dr. Doug Redelman, Sierra Cytometry

34. Human Sperm - PI - Sybr-Green I
live
inactive
active
dead
Data from Dr. Doug Redelman, Sierra Cytometry

35. Microbiology Detection of unknown organisms
Antibiotic sensitivity testing
Detection of Spores

36. Uptake of rhodamine 123 by M.luteus
Changes in light scattering behaviour and in the ability to accumulate Rhodamine 123 during resuscitation of a starved cultured of M. luteus. Cells were starved for 2.5 months, incubated with penicillin G for 10 hours, washed, and resuscitated in weak nutrient broth. Data represent a culture (A) immediately after the penicillin treatment, and (B) 2 days later.
Data from Dr. Hazel Davey

37. Mixed suspensions of bacteria Identification on scatter alone?BG
BG doublets
E.coli
Count
log SS
doublets ?
debris
BG spores
E.coli cells
debris
log FS
log FS
Light scatter signature of a mixture of B.subtilis
spores (BG) and E.coli cells.

38. Light Scatter of Bacterial Spores
B.anthracis SS
B.subtilis
irradiated B.anthracis FS
Light scatter signals from a mixture of live B.anthracis spores, live B. subtilis spores and gamma irradiated B. anthracis spores.

39. Nucleic Acid Content
Distinguish bacteria from particles of similar size
by their nucleic acid content
Fluorescent dyes
-must be relatively specific for nucleic acids
-must be fluorescent only when bound to
nucleic acids
Examples
DAPI
Hoechst 33342
cyanine dyes YoYo-1, YoPro-1, ToTo-1

40. Run on Coulter XL cytometer
mixture
Run on Coulter XL cytometer
mixture
Scatter BG
BG E.coli
Scatter
E.coli
Yo-Yo ex membrane impermeant so cells must be fixed.
Fix in ethOH - stain 5 minutes run flow
Spore coat difficult to get dyes in. Spores have less fluorescent than vegetative bacteria
Fluorescence
YoYo-1 stained mixture of 70% ethanol fixed
E.coli cells and B.subtilis (BG) spores.

41. Microbial Identification Using Antibodies
Enumeration & identification of target organisms in
mixed populations
Examples include:
Legionella spp. in water cooling towers
Cryptosporidium & Giardia in water reservoirs
Listeria monocytogenes in milk
E.coli O157:H7 in contaminated meat
Bacillus anthracis & Yersinia pestis biowarfare agents

42. Phenotyping - Immunophenotyping
Characterization of white blood cells
Identification of lymphocyte subsets

43. CELLULAR ANTIGENS Adhesion Receptors Metabolic T cells B Cells
cytokines
structure
enzymes
In the most recent leukocyte typing workshop over 160 antibodies were classified into defined groups. These antibodies bind to cytokines or chemokine receptors, sensory molecules, cytokine or chemokine ligands, metabolic proteins, adhesion molecules, enzymes, structural proteins and other important functional proteins within the cell. These antibodies can be used to identify specific cell subsets by their unique repertoire of molecular expression as well as their functional state using multiparameter flow cytometry.
Adhesion
Receptors
Metabolic
T cells
B Cells
courtesy of Jim Bender

44. Immunofluorescence staining
specific binding
nonspecific binding
Data from Dr. Carleton Stewart

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

46. Indirect staining Fluorescent probe attached to a 2nd antibody
Specific signal: strong, nd Ab/each 1st Ab; therefore dyes/site
Nonspecific binding: high
Data from Dr. Carleton Stewart

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

48. Three Color Lymphocyte Patterns
CD4
CD4
CD3
CD8
When more than two antibodies are combined, additional subsetting of the cells can be achieved. If one antibody is used to identify a specific lineage of cells, i.e. CD3, five subsets of T-cells are easily resolved, when CD4 vs C8 is displayed.
CD8
CD3
Data from Dr. Carleton Stewart

49. FOUR COLOR PATTERN CD56 CD8 CD4 CD3 CD3 CD3 CD8 CD4 CD4 CD56 CD56 CD8
As additional antibodies are combined, further subsetting can be achieved and heretofore unknown populations of cells discovered (CD3+CD4+CD8-CD56+). The increased dimensionality of data may make its visualization difficult.
CD8
CD4
CD4
CD56
CD56
CD8
Data from Dr. Carleton Stewart

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

51. Cellular Function Phagocytosis Killing index of phagocytes
Intracellular cytokines
Calcium flux
Oxidative burst
Membrane potential

52. Conclusions Many current research tools have clinical application
Frequently used in clinical trials and clinical research
Applications in veterinary medicine require
Cost reduction
Antibody specificity
Increased interest from veterinary researchers

53. Thank you for your attention
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