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 ©1990-2011

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

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

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+).

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

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

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)

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

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

Multicolor Analysis Roederer, et al

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

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

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

Analysis Logic Map Format

2:29 PM J.Paul Robinson, Purdue University - BMS 631 lectures ©1990-2010 J.Paul Robinson, Purdue University

Link to CDDB 2:29 PM J.Paul Robinson, Purdue University - BMS 631 lectures ©1990-2010 J.Paul Robinson, Purdue University