1. 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
2:29 PM
J.Paul Robinson, Purdue University - BMS 631 lectures ©

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

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

4. Typical immune screen for flow cytometry
A basic lymphocyte panel will most probably consist of 8 monoclonal antibodies:
CD3, CD4, CD8, CDl9, and CDl6/56
A control is added as part of the 8 antibody combination (anti-CD45/CD14). This is included to facilitate distinguishing lymphocytes from monocytes. If you were to run the above panel you would be able to identify the frequency of
T-cells (CD3+)
B-cells (CDl9+)
natural killer cells (CD3-CD16+CD56+)
T-helper-inducer cells (CD3+CD4+)
T-suppressor/cytotoxic cells (CD3+CD8+).

5. This is a subset of cells It is CD3+ CD56+
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
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

6. Clinical Cytometry
4-9 color phenotyping is current standard
Samples are run single tubes
Preparation is primarily manual
Analysis is mostly manual
Analysis is essentially the same as it was 20 years ago

7. Typical 4 Color Clinical Panel
1. CD3/CD14/DR/CD45 2. CD3/CD4/CD8/CD45 3. CD7/CD13/CD2/CD19 4. KAP/LAM/CD19/CD5 5. CD20/CD11c/CD22/CD23 6. CD38/CD10/CD19/CD34 7. CD57/CD56/CD33/CD3 8. CD11b/CD13/CD33/CD34 9. CD15/CD56/CD7/CD34
10. CD16/CD32/CD45/CD64
11. CD41/CD71/CD45/CD34
12. CD38/CD138/CD45/CD56
13. CD16/CD163/CD13/CD117
14. CD52/CD30/CD19/CD20
15. Isotype
16. Auto
17. Live Dead (Viability)

8. Clinical Sample – 17 Tube assay
Data kindly supplied by Dr. Paul Wallace

9. Similar situation with 9 color clinical samples
Data from Brent Wood publication

10. Multicolor Analysis
Roederer, et al

11. The power of cytomics 2:29:50 PM
High Throughput-High-Content Flow Cytometry
2:29:50 PM

12. Mass Cytometry - CyTOF 2:29:50 PM
High Throughput-High-Content Flow Cytometry

13. Very complex data sets 2:29:50 PM
High Throughput-High-Content Flow Cytometry

14. Analysis Logic Map Format

15. 2:29 PM
J.Paul Robinson, Purdue University - BMS 631 lectures © J.Paul Robinson, Purdue University

16. Link to CDDB
2:29 PM
J.Paul Robinson, Purdue University - BMS 631 lectures © J.Paul Robinson, Purdue University