Flow Cytometry for Everyone Else Susan McQuiston, J.D., MLS(ASCP), C.Cy.

At the end of this session, the participant will be able to: 1. Describe the components of a flow cytometer 2. Describe the gating for a multi-parametric population. 3. Interpret light scatter and fluorescence data plots

How does flow cytometry work? 1. Cells in suspension with fluorescent tags 2. Pass by laser(s)- interrogation point 1 4 3. The cell scatters light based on size and internal complexity 4. Fluorescent tags absorb laser energy and emit light 2 3

How does flow cytometry work? 5. Detectors convert light signal to electrical impulses 6 6. Software converts impulses to a display 5 7. Operator sets gates around populations 7 8. Software generates statistics 8

Let s take a closer look at the measurements and the data we are collecting by using peripheral blood from a healthy individual.

Light Scatter Internal complexity (refraction of cell components) Size (volume)

Fluorescence T lymphocytes express CD3 Anti-CD3*FITC B lymphocytes express CD19 Anti-CD19*PE

Fluorescence Intensity Compensation Detection wavelength for instrument FITC PE A = How much the FITC emission increases the PE signal intensity B= how much the PE emission increases the FITC signal Emission Wavelength

Strategy for Data Analysis Peripheral White Blood Cells FSC SSC lymphocytes T lymphocytes B lymphocytes

Light Scatter

CD19 PE Fluorescence From Light scatter GATE Negative For CD3 and CD 19 CD3 FITC What are these?

CD19: B Cells Statistics 9.26% NK cells 18.11% 55.35% CD3: T Cells

Quantitating Hematopoietic Stem Cells: Parameters 1. FSC: slightly larger than a lymphocyte 2. SSC: similar to a lymphocyte 3. CD45: very dim 4. CD34: positive 5. 7-AAD: viable cells are negative

SSC SSC Quantitating Hematopoietic Stem Cells 7-AAD CD45 C. Lemarie, How to Enumerate CD34+ cells, Flow Cytometry and Cell Analysis Workshop, ISCT Annual Meeting, 23 April 2014

SSC SSC Quantitating Hematopoietic Stem Cells CD34 CD45 C. Lemarie, How to Enumerate CD34+ cells, Flow Cytometry and Cell Analysis Workshop, ISCT Annual Meeting, 23 April 2014

SSC Quantitating Hematopoietic Stem Cells The percentage of cells in this gate are the hematopoietic stem cells FSC C. Lemarie, How to Enumerate CD34+ cells, Flow Cytometry and Cell Analysis Workshop, ISCT Annual Meeting, 23 April 2014

Lymphocytic Leukemia

Lymphocytic Leukemia Some basic marker staining- CD19 positive: B cell receptor CD22 weak positive= lack of surface immunoglobulin (excludes normal B cells) CD5/CD23 positive= rule out Mantle Cell Lymphoma CD7 negative= definitely not a T cell CLL Kappa Light chain positive= monoclonal expression indicative of CLL

Lymphocytic Leukemia http://www.scielo.br/scielo.php?script=sci_arttext&pid=s0102-86502005000700011

Lambda Lymphocytic Leukemia http://www.scielo.br/scielo.php?script=sci_arttext&pid=s0102-86502005000700011

B Cell Lymphocytic Leukemia CBC: Increased lymphocytes and smudge cells FLOW CYTOMETRY: CD 19 positive= B cell receptor CD22 weak positive= lack of surface immunoglobulin (excludes normal B cells) CD5/ CD23 positive= It s CLL, not Mantle Cell Lymphoma CD7 negative= it s not a T cell Kappa light chain positive= monoclonal expression

Bone Marrow SSC SSC FSC CD 45

Review 1. Describe the components of a flow cytometer Optics: Laser and Detectors (PMT) Fluidics: to carry cells and line them up in front of the laser Electronics: detector output to software visualization 2. Describe the gating for a multi-parametric population. Gate using light scatter ( T cells and B cells in peripheral blood Gate using a fluorescent antibody to a shared marker 3. Interpret light scatter and fluorescence data plots T cells and B cells CD34 quantitation Chronic lymphocytic leukemia

References Flow Cytometry in Clinical Diagnosis, Carey 2007 Practical Flow Cytometry, Shapiro 2003 Cytometry, Part A (research) & Part B (clinical) https://www.bdbiosciences.com/applications/ C. Lemarie, How to Enumerate CD34+ cells, Flow Cytometry and Cell Analysis Workshop, ISCT Annual Meeting, 23 April 2014 Blood April 15, 2008 vol. 111 no. 8 3941-3967 C. Lemarie, How to Enumerate CD34+ cells, Flow Cytometry and Cell Analysis Workshop, ISCT Annual Meeting, 23 April 2014 https://www.researchgate.net/figure/7158772_fig1_figure-2-bcell-developmentstages-of-b-cell-

Where you can learn more: Websites Becton Dickinson: https://www.bdbiosciences.com/us/s/flowcytometry?gclid=cjxhy9qa68scfqraqod1x0csa International Clinical Cytometry Society: http://www.cytometry.org/web/index.php International Society for Advancement of Cytometry: http://isac-net.org/ Continuing Education CACMLE: Principles and Clinical Applications of Clinical Cytometry Michigan State University: online courses, one week lab experience

Thank you! Questions?